Proteome Analysis of Interleukin-1␤–Induced Changes
in Protein Expression in Rat Islets of Langerhans
P. Mose Larsen,1 S.J. Fey,1 M.R. Larsen,2 A. Nawrocki,1 H.U. Andersen,3 H. Kähler,3 C. Heilmann,3
M.C. Voss,3 P. Roepstorff,2 F. Pociot,3 A.E. Karlsen,3 and J. Nerup3
The intracellular molecular events involved in the ␤-cell
death process are complex but poorly understood. Cytokines, e.g., interleukin (IL)-1␤, may play a crucial role
in inducing this process. Protein synthesis is necessary
for the deleterious effect of IL-1, and induction of both
protective and deleterious proteins has been described.
To characterize the rather complex pattern of islet
protein expression in rat islets in response to IL-1, we
have attempted to identify proteins of altered expression level after IL-1 exposure by 2D gel electrophoresis
and mass spectrometry. Of 105 significantly changed
(i.e., up- or downregulated or de novo–induced) protein
spots, we obtained positive protein identification for 60
protein spots. The 60 identifications corresponded to 57
different proteins. Of these, 10 proteins were present in
two to four spots, suggesting that posttranslatory modifications had occurred. In addition, 11 spots contained
more than one protein. The proteins could be classified
according to their function into the following groups:
1) energy transduction; 2) glycolytic pathway; 3) protein synthesis, chaperones, and protein folding; and
4) signal transduction, regulation, differentiation, and
apoptosis. In conclusion, valuable information about
the molecular mechanisms involved in cytokine-mediated ␤-cell destruction was obtained by this approach.
Diabetes 50:1056 –1063, 2001
elective ␤-cell destruction accompanied by
mononuclear cell infiltration of the islets of Langerhans (insulitis) is the hallmark of recent-onset
type 1 diabetes in humans (1,2) and animal models of type 1 diabetes (e.g., the NOD mouse [3] and the BB
rat [4]). Cytokines and cytotoxic T-cells are likely to be the
most important mediators of selective ␤-cell destruction
(rev. in 5–7). The intracellular molecular events involved
in the ␤-cell death process are complex and poorly understood. Cytokines (interleukin [IL]-1 in particular) induce
the synthesis of the enzyme inducible nitric oxide synthase
S
From the 1Center for Proteome Analysis and the 2Institute for Biochemistry
and Molecular Biology, University of Southern Denmark, Odense; and the
3
Steno Diabetes Center, Gentofte, Denmark.
Address correspondence and reprint requests to Dr. Peter Mose Larsen,
Center for Proteome Analysis, Forskerparken Fyn, Odense, Denmark. E-mail:
nerup@pres.dk.
Received for publication 6 September 2000 and accepted in revised form 5
February 2001.
BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium;
HBSS, Hanks’ balanced salt solution; HSP, heat shock protein; IEF, isoelectric
focusing; IL, interleukin; iNOS, inducible nitric oxide synthase; MALDI, matrixassisted laser desorption/ionization; MS, mass spectrometry; NEPHGE, nonequilibrium pH-gradient electrophoresis; NHS, normal human serum; NO, nitric oxide;
pI, isoelectric point; SOD, super oxide dismutase; TCA, trichloroacetic acid.
1056
(iNOS) (rev. in 8) and the expression of Fas (9) in ␤-cells.
analogs, like NMMA, which prevent nitric oxide
(NO) production from iNOS, partially protect ␤-cells from
cytokine toxicity (rev. in 8), and hyperexpression of the
antiapoptotic molecule Bcl-2 (10) or scavengers of oxygen
free radicals (e.g., catalase, glutathione peroxidase, and
Cu/Zn super oxide dismutase [SOD] [11]) markedly reduce
the destruction of ␤-cells induced by cytokines.
Protein synthesis inhibitors (e.g., cycloheximide) effectively protect IL-1– exposed islets from destruction (12).
Hence, protein synthesis is necessary for the deleterious
effect of IL-1. Previous studies have shown that IL-1
induces the synthesis of members of the heat shock
protein (HSP) family, like heme oxygenase (13) and HSPs
70 and 90 (14,15), and hyperexpression of HSPs in islets is
partially protective against cytokine-induced ␤-cell destruction (16). Furthermore, it has been reported that IL-1
may induce the synthesis of unknown proteins with molecular weights of 45, 50, 75, 85, 95, and 120 kDa (17). It has
previously been shown that rat islets exposed to IL-1
release NO into the culture media (18). iNOS has been
cloned from islets (19) in which it has been shown to be
inducible in ␤-cells only (20). We have further shown that
IL-1 also upregulates IL-1 converting enzyme mRNA transcription (21) in rat islets. Also, SOD is shown to be
upregulated in islets by cytokines (15,22).
Based on ␤-cell–selective toxic effects, we hypothesized
that IL-1 induces a rather complex pattern of both protective and deleterious events and mechanisms in islets cells,
and that in ␤-cells the deleterious events seem to prevail
(23). We further suggested that this might be reflected at
the level of islet protein expression (24). To examine this
hypothesis, we used 2D gel electrophoresis to produce a
database of rat islet proteins containing about 2,200 protein spots characterized by molecular weight and isoelectric point (pI). The data presented here provide the first
global assessment of the IL-1–mediated ␤-cell– damaging
processes at the protein level. We could demonstrate that
IL-1 exposure of the islets in culture resulted in reproducible and statistically significant modulation of protein
expression levels or de novo synthesis of 105 of these
proteins. A total of 52 proteins were upregulated, 47 downregulated, and 6 synthesized de novo (25).
The aim of the present study was to positively identify
these 105 proteins in order to better understand ␤-cell
destruction and type 1 diabetes at the molecular level.
L-Arginine
RESEARCH DESIGN AND METHODS
Reagents. Dulbecco’s modified Eagle’s medium (DMEM), RPMI-1640, and
Hanks’ balanced salt solution (HBSS) were purchased from Life Technologies
DIABETES, VOL. 50, MAY 2001
P.M. LARSEN AND ASSOCIATES
(Paisley, Scotland). RPMI-1640 was supplemented with 20 mmol/l HEPES
buffer, 100,000 IU/l penicillin, and 100 mg/l streptomycin. Authentic recombinant human IL-1 was provided by Novo Nordisk (Bagsværd, Denmark). The
specific activity was 400 U/ng. The following other reagents were used:
2-mercaptoethanol, bovine serum albumin (BSA), Tris-HCl, Tris-base, glycine,
(Sigma, St. Louis, MO); trichloroacetic acid (TCA), phosphoric acid, NaOH,
glycerol, n-butanol, Bromophenol blue (Merck, Darmstadt, Germany); 35Smethionine (SJ 204, specific activity: ⬎1,000 Ci/mmol, containing 0.1% 2-mercapthoethanol), Amplify (Amersham International, Amersham, U.K.); filters
(HAWP 0.25-mm pore size; Millipore, Boston, MA); RNase A, DNase I (Worthington, Freehold, NJ); urea (ultra pure) (Schwarz/Mann, Cambridge, MA);
acrylamide, N,N1-methylenebisacrylamide, TEMED, ammonium persulfate
(Bio-Rad, Richmond, CA); carrier ampholytes (pH 5–7, pH 3.5–10, pH 7–9, pH
8 –9.5) (Pharmacia, Uppsala, Sweden); Nonidet P-40 (BDH, Poole, UK); carrier
ampholytes (pH 5–7 and sodium dodecyl sulfate) (Serva, Heidelberg, Germany); agarose (Litex, Copenhagen); ethanol (absolute 96%) (Danish Distillers,
Aalborg, Denmark); methanol (Prolabo, Brione Le Blanc, France); acetic acid
(technical quality, 99% glacial) (Bie & Berntsen, Århus, Denmark); and X-ray
film (Curix RP-2; AGFA).
Islet isolation and culture. For the database and assay variation experiments, 12 different islet isolations were performed, 10 for the databases, 1 for
intra-assay, and 1 for interassay analysis (24). For the protein identification
studies, additional islet isolations were performed (a total of ⬃200,000 islets).
Islets from pancreata of 4-day-old inbred Wistar Furth rats (Møllegård, Lille
Skensved, Denmark) were isolated after collagenase digestion (26). After a
preculture period of 4 days in RPMI-1640 plus 10% fetal calf serum, sets of 150
islets were incubated for 24 h in 300 ␮l RPMI-1640 plus 0.5% normal human
serum (NHS) for labeling with 35S-methionine (tracer islets) (24). The remaining islets were used for preparatory gels for protein identification. In parallel
experiments, 150 islets were incubated for 24 h in 300 ␮l RPMI-1640 plus 0.5%
NSH with or without the addition of 150 pg/ml IL-1 for functional studies of
NO and insulin secretion. To reduce variation, the same batches of fetal calf
serum and NHS were used throughout the experiment.
Islet protein labeling. After 24 h in culture, the 150 islets were harvested,
washed twice in HBSS, and labeled for 4 h in 200 ␮l methionine-free Dulbecco’s modified Eagle’s medium (DMEM) with 10% NHS dialysed for amino
acids and 200 ␮Ci 35S-methionine (24). To reduce variation, the same batch of
NHS has been used throughout the experiment. To eliminate 2-mercaptoethanol, 35S-methionine was freeze-dried for at least 4 h before labeling. After
labeling, islets were washed thrice in HBSS, pelleted, and frozen at ⫺80°C.
Sample preparation. The frozen islets were resuspended in 100 ␮l DNase/
RNase A solution and lysed by freeze-thawing twice. After the second thawing,
they were left on ice for 30 min for the digestion of nucleic acids. The lysed
sample was then freeze-dried overnight. The samples were dissolved by
shaking in 120 ␮l lysis buffer (8.5 mol/l urea, 2% Nonidet P-40, 5% 2-mercaptoethanol, and 2% carrier ampholytes, pH range 7–9) for a minimum of 4 h.
Determination of 35S-methionine incorporation. The amount of 35Smethionine incorporation was quantitated in duplicate by adding 10 ␮l BSA
(0.2 ␮g/ml H2O) as a carrier to 5 ␮l of a 1:10 dilution of each sample, followed
by 0.5 ml of 10% TCA. This was left to precipitate for 30 min at 4°C before
being filtered through 0.25-␮m filters. The HAWP filters were dried and placed
into scintillation liquid for counting.
2D gel electrophoresis. The procedure was essentially performed as previously described (27–29). Briefly, first-dimensional gels contained 4% acrylamide, 0.25% bisacrylamide, and carrier ampholytes (the actual ratio
depending on the batch) and were 175 mm long and 1.55 mm in diameter. An
equal number of counts (106 cpm) of each sample was applied to the gels. In
case of lower amounts of radioactivity, it was necessary to regulate the
exposure time of the gel so that comparable total optical densities were
obtained. The samples were analyzed on both isoelectric focusing (IEF) (pH
3.5–7) and nonequilibrium pH-gradient electrophoresis (NEPHGE) (pH 6.5–
10.5) gels. IEF gels were prefocused for ⬃4 h at 140 ␮A/gel (limiting current);
the sample was then applied and focused for 18 h at 1,200 V (limiting voltage).
NEPHGE gels were focused for ⬃6.5 h using 140 ␮A/gel and 1,200 V as the
limiting parameters. Second-dimension gels, 1 ⫻ 200 ⫻ 185 mm, contained
either 15% acrylamide and 0.075% Bis or 10% acrylamide and 0.05% Bis, and
they were run overnight. This separation protocol was optimized for hydrophilic proteins; thus, a detailed characterization of hydrophobic (membrane)
proteins is not possible. After electrophoresis, the gels were fixed in 45%
methanol and 7.5% acetic acid for 45 min and treated for fluorography with
Amplify for 45 min before being dried. The gels were placed in contact with
X-ray films and exposed at ⫺70°C for 1– 40 days. Each gel was exposed for at
least three time periods to compensate for the lack of dynamic range of X-ray
films.
Determination of Mr and pI. Mr and pI for individual proteins on the gels
was determined by the use of internal standards. Theoretical Mr and pI were
DIABETES, VOL. 50, MAY 2001
calculated using the Compute pI/Mw tool at the ExPASy Molecular Biology
Server (www.expasy.ch/tools/pi_tool.html).
Protein characterization. Preparatory 2D gels were produced from a pool
of ⬃200,000 neonatal WF islets that were isolated, prepared, and separated on
gels as described above. For localization of the spots, radioactively labeled
tracer islets were mixed with the nonlabeled islets. Because initial attempts to
identify the proteins in the gel resulted in very few positive identifications by
direct microsequencing, the method of choice became mass spectrometry
(MS).
Protein identification by MS. Briefly, protein spots of interest were
obtained by cutting them from the dried gel using a scalpel. A total of 103
spots could technically be cut from the gels for analysis. The proteins were
enzymatically digested in the gel as described (30,31), with minor modifications (32). The excised gel plugs were washed in 50 mmol/l NH4HCO3/
acetonitrile (60/40) and dried by vacuum centrifugation. Modified porcine
trypsin (12 ng/␮l, sequencing grade; Promega) in digestion buffer (50 mmol/l
NH4HCO3) was added to the dry gel pieces and incubated on ice for 1 h for
reswelling. After removing the supernatant, 20 – 40 ␮l digestion buffer was
added and the digestion was continued at 37°C for 4 –18 h. The peptides were
extracted as described (31) and dried in a vacuum centrifuge. The residue was
dissolved in 5% formic acid and analyzed by matrix-assisted laser desorption/
ionization (MALDI) MS. Delayed extraction MALDI mass spectra of the
peptide mixtures resulting from in-gel digestion were acquired using a
PerSeptive Biosystems Voyager Elite reflector time-of-flight mass spectrometer (Perseptive Biosystems, Framingham, MA). Samples were prepared using
␣a-cyano-4-hydroxy cinnamic acid as matrix. When appropriate, nitrocellulose
was mixed with the matrix (33). Protein identification was performed by
searching the peptide-mass maps in a comprehensive nonredundant protein
sequence database (NRDB; European Bioinformatics Institute, Hinxton, U.K.)
using the PeptideSearch software (34) further developed at EMBL (Heidelberg, Germany). The protein identifications were examined using the “second
pass search” feature of the software and critical evaluation of the peptide
mass map as described (35).
In cases in which protein spots to be examined by MS were present at low
abundance in the 2D gels of islet material, and in cases in which results were
ambiguous, confirmatory MS analysis was performed on protein spots with
the same coordinates (Mr and pI) from large batches of rat insulinoma (RIN)
cells treated as described for islets.
The following protein databases were searched for matches: SWISS-PROT,
PIR, National Institutes of Health, and Genebank.
RESULTS
As might have been anticipated from the available amount
of protein in each protein spot, which was estimated to
range from ⬍50 to 500 ng with very large variation
from spot to spot, initial attempts to identify the proteins
by microsequencing were of little use (data not shown).
However, using MS, 57 positive protein identifications
were obtained (Table 1). Six proteins (GAPDH, pyruvate
kinase M, ATP synthase regulatory subunit A, acetyl-CoA
acetyl transferase, mortalin [GRP75] and protein disulfide
isomerase ER60) were present in two protein spots, three
proteins (pyruvate kinase M2, glutamate dehydrogenase
and neuroendocrine convertase two precursor) were
present in three spots, and one protein (glucose-regulated
protein precursor [GRP78]) was present in four spots,
suggesting that these proteins may be posttranslatory
modified. Nine protein spots (IEF 28, 329, 614, and 908 and
NEPHGE 130, 174, 182, 231, and 269) contained two
identified proteins, and two spots (IEF 387 and NEPHGE
102) contained three identified proteins. Neither positive
protein identification nor useful mass spectra could be
obtained from the remaining 43 protein spots.
For proteins with observed Mr’s ⬎100 kDa (spots IEF
11, 85, 173, 186, 187, 194, 201, and 265), the calculated Mr’s
are considerably lower (20 –50%). This may be explained
by relatively imprecise Mr determination of high–molecular weight proteins on the gels. Only minor inconsistencies
are found between observed and theoretical Mr’s for
proteins with molecular weights ⬍100 kDa, with two
1057
PROTEOME ANALYSIS OF RAT ISLETS
TABLE 1
Known and putative functions of identified proteins
Gel spot no.
% IOD
IOD ratio
I. Energy transduction and redox potentials
NEPHGE 171/176† 0.41/0.91
0.21/0.21
Protein
ATP synthase regulatory
subunit (A)
NEPHGE 174
0.27
0.43
Rat mitochondrial H⫹-ATP
synthase alpha subunit
IEF 614
0.93
0.27
ATP synthase catalytic
subunit (B)
NEPHGE 306
0.29
0.09
Adenylate kinase isoenzyme 2 (mitochondrial)
IEF 265
0.05
3.31
Transitional endoplasmic
reticulum ATPase
IEF 25
0.16
1.79
Vacuolar ATP synthase
subunit B, brain isoform,
bovine
NEPHGE 102
0.26
0.45
5-aminoimidazole-4-carboxamid ribonucleotide
formyl transferase IMP
cyclohydrolase
NEPHGE 227/231
0.34/0.19
0.29/0.41
Acetyl-CoA acetyl transferase (ACAT2)
NEPHGE 182
1.78
0.55
L-3-hydroxyacyl-CoA dehydrogenase
NEPHGE 296†
5.80
0.07
NADH-cytochrome B5 reductase
IEF 329
0.10
2.23
NADPH-cytochrome P450
reductase
NEPHGE 231
0.19
0.41
Creatin kinase (ubiquitous
mitochondrial form)
NEPHGE 18/174/182 3.57/0.27/1.78 0.27/0.43/0.55 Glutamate dehydrogenase
(GDH)
NEPHGE 130
0.24
0.45
Methylmalonate-semialdehyde dehydrogenase
II. Glycolytic pathway
NEPHGE 169
0.28
2.16
6-phospho fructo-2-kinase
NEPHGE 668†
DN*
Fructose 1,6-biphosphate
aldolase A
NEPHGE 334
0.57
0.11
Triose phosphate isomerase (TPI1)
NEPHGE 17†/269
0.40/0.15
5.09/9.04
Glyceraldehyde-3-p-dehydrogenase (GAPDH)
NEPHGE 203
1.64
1.51
Phospho-glycerate kinase
IEF 908†
0.08
0.27
Phospho-glycerate mutase
(PGAM), brain form
IEF 561
0.12
2.52
Enolase ␣/1
NEPHGE 102/130
0.26/0.24
0.45/0.45
Pyruvate kinase M (processed pseudogene)
NEPHGE 1/123/129 0.43/0.72/0.62 0.17/0.29/0.32 Pyruvate kinase M2, early
fetal tissue
IEF 85
0.004
4.21
Pyruvate carboxylase
III. Protein synthesis, chaperones, and protein folding
IEF 11
0.03
2.34
Elongation factor 2 (EF2,
polypeptidyl–tRNA
translocase)
IEF 28†
0.11
2.65
Heterogeneous nuclear
ribonucleoprotein
K/ROK
NEPHGE 156†
0.01
3.22
Polypyrimidine tract-binding protein (PTB)
IEF 173
0.05
2.47
Major vault protein (RNP
protein for nucleocytoplasmic transport)
Database
accession no.
Mr
Theoretical
Mr
pI
Theoretical
pI
P15999
52.8/52.6
58.8/55.3
8.2/8.0
9.2/8.3
J05266
53.8
58.8
7.9
9.2
P10719
52.9
56.4/51.7
4.8
5.2/5.0
P29410
35.7
26.2
8.1
6.4
P46462
120.0
89.3
5.0
5.1
P31408
54.2
56.6
5.5
5.7
BAA22837
63.6
64.2
7.3
6.7
P17764
43.9/44.4
44.7/41.4
8.4/8.3
8.9/8.4
ADD42162
54.1
34.4
7.6
9.4
P20070
36.2
34.0
8.3
8.6
P00388
72.9
76.8
5.3
5.3
P25809
44.3
47.0/43.1
8.3
8.7/7.8
P10860
36.4/53.8/54.1 61.4/58.8/55.9 8.4/7.9/7.6
8.1/9.2/6.7
Q02253
55.7
57.8
8.1
8.5
P07953
P05065
55.6
42.8
54.6
39.2
8.2
8.5
6.3
8.4
P48500
30.9
26.8
8.2
6.5
P04797
40.0/40.0
35.7
8.3/8.0
8.4
P16617
P25113
44.4
25.8
44.4
28.5
8.2
6.3
7.5
6.2
P04764
M24361
49.3
63.6/55.7
47.0
141.4
6.0
7.3/8.1
6.2
4.9
P11981
58.8/57.0/57.6 57.6
8.3/8.2/7.7
7.4
P52873
164.1
127.4
6.3
6.1
P05197
118.7
95.3
6.6
6.4
S41495
65.8
60.0
5.1
5.4
Q00438
55.6
59.3
8.7
9.2
Q62667
136.7
98.5
5.4
5.7
Continued on following page
1058
DIABETES, VOL. 50, MAY 2001
P.M. LARSEN AND ASSOCIATES
TABLE 1
Continued
Gel spot no.
IEF 310
% IOD
0.10
IOD ratio
Database
accession no.
Protein
1.88
Glycyl-tRNA synthetase (human)
IEF387
0.15
2.41
T-complex protein 1
(␥-subunit), human/mouse
IEF 506
0.17
2.34
T-complex protein 1
(ε-subunit), mouse
NEPHGE 102
0.26
0.45
T-complex protein 1
(␨-subunit), mouse
NEPHGE 326
0.09
0.17
Caldesmon, human
IEF 895†
0.11
0.69
Tropomyosin NM4 (TMP-␥)
NEPHGE 269
0.15
9.04
Annexin II
IEF 201
0.27
2.48
Calnexin
IEF 187†
0.02
6.19
Ischemia-responsive protein
94 kDa (irp94)
IEF 267
1.69
2.12
HSP90-␤ (HSP84)
IEF 425†
0.55
2.09
HSP71c (heat shock cognate
71-kDa protein)
IEF 186
0.01
22.00
HSP70KD protein AGP-2
(mouse)
IEF 507†
0.16
2.72
Mitochondrial matrix protein p1 (HSP60), mouse
IEF 329/339
0.10/0.18
2.23/0.16
Glucose-regulated protein
IEF 344/347
0.09/0.46
0.44/4.05
precursor-HSPA5 (GRP78)
IEF 330/340†
0.08/0.16
2.74/0.32
Mortalin (GRP75)
IEF 483/484†
0.17/0.17
1.72/0.27
Protein disulfide isomerase
(PDI) ER60 (GRP58)
IEF 614
0.93
0.27
Probable protein disulfide
isomerase P5
IEF 908†
0.08
0.27
ER protein (ERP31 precursor to ERP29)
IEF 387
0.15
2.41
Coatomer (␦-subunit), bovine/human
IEF 194
0.02
3.31
Ubiquitin COOH-terminal
hydrolase T, mouse
IV. Signal transduction, regulation, differentiation and apoptosis
IEF 941
0.29
0.08
Phosphatidylethanolaminebinding protein (P23K)
IEF 655
0.04
1.85
Lamin A (split product)
IEF 28†
0.11
2.65
Lamin B1
IEF 759
0.12
2.40
TGF-␤ receptor interacting
protein 1 (TGFrip)
IEF 949
0.17
0.21
14-3-3 protein-ε-isoform
IEF 387
0.15
2.41
Turned on after division
(TOAD-64)
IEF 436/441/442 0.31/0.16/0.16 0.38/0.22/0.16 Neuroendocrine convertase
2 precurser (NEC2)
IEF 665
0.09
0.33
Metastasis associated protein (MTA-1)
NEPHGE 298
0.70
24.45
Galectin-3, MAC-2 antigen
Mr
Theoretical
Mr
pI
Theoretical
pI
P41250
69.4
77.5
5.7
5.9
P80318
64.3
60.6
6.1
6.3
P80316
60.0
59.6
5.4
5.7
P80317
63.6
58.0
7.3
6.6
Q05682
L24777
Q07936
P35565
AAC27937
34.5
28.9
39.9
143.6
152.1
—
28.7
38.5
—
94.1
8.6
4.3
8.0
4.1
5.0
—
4.8
7.5
—
5.1
P34058
P08109
92.5
67.2
83.2
70.9
4.8
5.2
5.1
5.4
Q61316
152.1
94.1
5.0
5.2
P19226
59.9
61.0
5.3
5.9
P06761
72.9/72.6
5.3/5.1
P48721
P11598
68.8/71.3
31.2/59.2
72.3
73.6/77.7
68.8/73.9
56.6
5.4/5.2
5.7/5.9
5.1
5.0/4.6
5.5/6.0
5.9
Q63081
52.9
47.2
4.8
5.0
P52555
25.8
28.6
6.3
6.2
P53619
64.3
57.3
6.1
5.9
P56399
139.3
95.8
4.7
4.9
P31044
22.7
20.8
5.2
5.5
P48679
U72353
U36764
41.4
65.8
37.1
74.3
66.6
36.5
6.2
5.1
5.3
6.5
5.2
5.4
U53882
P47942
26.8
64.3
29.1
62.3
4.5
6.1
4.6
6.0
P28841
66.4/66.8/66.9
70.8
4.8/4.6/4.5
5.9
Q62599
42.2
79.4
5.8
9.3
P08699
36.4
27.1
8.3
8.6
The proteins in this table are arranged in the order that they are referred to in DISCUSSION. The gel spot numbers refer to numbers in the rat
islet protein database, published by Andersen et al. (25). The % IOD represents the average of the % IODs assigned to the same spot in five
independent experiments. The IOD ratio expresses the % IOD of a protein spot in 2D gels of IL-1␦ exposed rat islets/% IOD of the same spot
in 2D gels of rat islets not exposed to IL-1␦. Hence, a % IOD ratio above 1 indicates that the spot is upregulated by IL-1␦ (25). *DN, a protein
induced to synthesis de novo. Protein spots related by posttranslational modification are given in the same row. Theoretical pIs and molecular
weights have been calculated for the mature proteins where applicable. Proteins previously shown to be altered by IL-1 in an NO-dependent
manner (60) are marked with †.
exceptions (spots IEF 665 and NEPHGE 306). These
proteins also show major inconsistencies between observed and theoretical pI values. If identifications are
correct, this suggests that these proteins have been subject to posttranslational modification. Where available
from the Compute pI/Mw tool at the ExPASy Molecular
DIABETES, VOL. 50, MAY 2001
Biology Server, we have included the theoretical calculated values for both the native and posttranslationally
modified protein.
Some protein spots (IEF 25, IEF 186, IEF 194, IEF 310,
IEF 387, IEF 506, IEF 507, and NEPHGE 102) contained
proteins that were identified through homology to other
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PROTEOME ANALYSIS OF RAT ISLETS
species (Table 1); at the time of the database search, their
rat sequences were not entered.
Hence, the success rate for MALDI MS identification of
proteins was 58% for positive identification. It’s beyond
the space available to the present article to describe
the functions and possible importance for type 1 diabetes
pathogenesis for each of the identified proteins in detail.
However, in Table 1, the identified proteins are assigned to
broad classes according to their known or putative functions: 1) energy transduction and redox potentials; 2) glycolytic pathway; 3) protein synthesis, chaperones, and
protein folding; and 4) signal transduction, regulation,
differentiation, and apoptosis.
DISCUSSION
A success rate of 58% for positive identification by MS is
acceptable considering the rather minute amounts of islet
tissue on average in the low nanogram amounts. It demonstrates that MS can be used for large-scale screening of
protein identities. The high-resolution 2D gel technology
can effectively separate proteins and offers the possibility
of relative quantification of changes in protein expression
as well as identification of certain posttranslatory protein
modifications (e.g., induced by cleavage and/or chemically
like phosphorylation) induced by interventions. Combined
2D gel and MS technology may be useful tools for dynamic
studies of molecular processes underlying complex disease processes, like the ones leading to ␤-cell destruction
and type 1 diabetes.
Messenger RNA differential display techniques have
been applied to various target tissues, including islet tissue, in an attempt to identify differences in specific mRNA
expression and genes of relevance for type 1 diabetes as
well as for type 2 diabetes (36 –38). However, in contrast to
mRNA (cDNA) expression studies, protein expression
studies allow assessment of posttranslational modifications. Posttranslational modifications are often necessary
for protein function and, hence, may also be of pathogenetic relevance. Thus, for the time being, the combination
of high-resolution 2D gel technology and MS seems to be
the method of choice for investigating disease processes.
Several studies have documented the deleterious functional and morphologic effects of IL-1␤ on isolated rat
islets in vitro (5–7) and the finding that both protein
synthesis and suppression (25) take place in islets exposed
to IL-1 in vitro. On the basis of these and other observations, we proposed (5) that cytokines, and IL-1 in particular, were responsible for initiation of the processes
producing spontaneous type 1 diabetes in animals and
humans. We further hypothesized that islet exposure to
cytokines would induce a complex response pattern in
islets and ␤-cells comprising protective (e.g., upregulation
of stress proteins) as well as deleterious (e.g., iNOS induction and NO production) events. The findings presented
here support this hypothesis. Islet function as measured
by NO production and insulin release was impaired by
exposure to IL-1 (data not shown) in complete accordance
with previous studies from our group (24). As evidenced in
Table 1, expression of several proteins of known or
putative importance for glycolysis and mitochondrial energy production, gene transcription, protein synthesis, and
apoptosis are affected by cytokine exposure.
1060
In Table 1, we have grouped the identified proteins
according to the cellular pathways in which they may be
involved. In the following sections, only some of these
findings are discussed in relation to previous observations
of cytokine effects on islets. Proteins that were identified
in this study are indicated by italic letters.
Energy transduction and redox potentials. It is well
documented that IL-1 has an inhibitory effect on mitochondrial energy production in islets (39,40). Here, two components of the five, which form the mitochondrial ATP synthase, were identified: the regulatory subunit A and the
catalytic subunit B. Both are strongly downregulated in response to IL-1 (by the ratios of 0.21 and 0.29, respectively).
Adenylate kinase, another mitochondrial enzyme (outer
compartment), which can interconvert ATP and AMP to
ADP, was also strongly downregulated (integrated optical
density ratio 0.09). Thus, there appears to be less ATP synthesized or available in the mitochondria, in accordance
with previous observations (41,42).
In contrast, both the ATPase of the transitional
endoplasmic reticulum and the vacuolar ATPase are
upregulated.
Further, acetyl-CoA acetyl transferase and 3-hydroxyacyl-CoA dehydrogenase, which are both involved in fatty
acid oxidation, were found to be downregulated.
NADH cytochrome B5 reductase, which is located to the
cytoplasmic side of the endoplasmic reticulum and mitochondrial outer membranes, is known to be involved in the
desaturation and elongation of fatty acids, and cholesterol
biosynthesis was shown to be strongly downregulated.
The glycolytic pathway. Whereas cytokines have been
shown to decrease the mitochondrial oxidation of glucose,
the effect on the glycolytic pathway is not clear (6). It has
been described that cytokines neither decrease function of
the glycolytic pathway nor affect the activity of several key
glycolytic enzymes (41,42). How this observation correlates to the changed expression of nine enzymes in the
glycolytic pathway in response to IL-1 found in the present
study remains to be clarified (Table 1).
Several of the enzymes involved in substrate metabolism have been demonstrated to have their synthesis and
activity regulated by cellular ATP levels (42). This may in
part explain the observed changes in expression levels of
proteins involved in the metabolic pathways.
Protein synthesis, chaperones, and protein folding.
Several studies have shown IL-1 to influence gene transcription and protein synthesis (12,14,25,43) and have
shown IL-1 to cause a fall in the overall rate of protein synthesis and of preproinsulin biosynthesis in particular (44).
Three separate components of the TCP-1 T-complex
were identified (gamma, epsilon, and zeta). TCP-1 (CCT)
is a ring shaped 950-kDa complex of eight polypeptides
encoded by different genes. This complex functions as a
type II chaperone and works specifically to fold and produce native actin, tubulin, and probably a few other proteins (45).
Formation and maintenance of the actin cytoskeleton is
important, and proteins with functions in this context
were identified. Caldesmon is a protein that binds both
actin and myosin and promotes the binding of yet another
protein detected here, tropomyosin gamma, to the actinomyosin bundles. Annexin II has been shown to interact
DIABETES, VOL. 50, MAY 2001
P.M. LARSEN AND ASSOCIATES
with CD44 (one of the major cell surface receptors for
hyaluronic acid) in the formation of cholesterol-rich lipid
rafts (46). Annexin II is located on the cytoplasmic side of
these rafts and appears to play a role in the binding to the
actin bundles.
IL-1–induced altered expression levels of seven members of the HSP family were identified. HSPs are well established as molecular chaperones, with functions within
folding/unfolding, assembly/disassembly, and transport of
protein (47). Furthermore, upregulated HSP70 expression
has been demonstrated to prevent the inhibitory effect of
IL-1 on islet insulin secretion and NO-induced mitochondrial impairment (48,49). In the diabetes-prone BB rat,
insufficient HSP70 expression has been correlated to high
vulnerability to NO and oxygen radicals (50). The newly
described irp94 (ischemia-responsive protein 94 kDa), a
member of the HPS110 family, which is believed to be a
homologue of the mouse and human apg-2 protein, is upregulated under ischemic stress (51,52). This protein was
found to be strongly upregulated by IL-1 in the rat islets.
Irp94 is not involved in the heat shock signaling mechanism, but is involved in neuronal stress responses following transient forebrain ischemia. Several genes and proteins are involved (including irp94), and it has been
suggested that some of them may have a deleterious or
protective role in the support of neuronal survival. (52). It
is tempting to speculate that irp94 may play similar roles
in the ␤-cell.
Signal transduction, regulation, differentiation, and
apoptosis. Different cytokines may induce ␤-cell death
by different interacting pathways. Thus, the decreased
amounts of phosphatidylethanol-amine-binding protein
in IL-1– exposed islets may result in an increased sensitivity to tumor necrosis factor-␣ (53). Several studies have
demonstrated that one IL-1–induced death pathway in
islets and ␤-cells is mediated through apoptosis (54 –56).
This is in accordance with our finding of altered expression levels of several proteins involved in this complex
pathway, e.g., lamins A and B (rev. in 57,58) and TGF-␤
receptor interacting protein (59).
The overall picture is complex and reflects the range of
cellular responses to the IL-1 challenge. Thus, we do not
know which protein changes may be considered “primary”
or “secondary” in importance, time, and sequence. Furthermore, it has been shown that IL-1 may induce both
NO-dependent as well as NO-independent ␤-cell impairment (8,24). In this context, we have recently demonstrated that of the 105 protein spots, which changed
expression levels in response to IL-1 (25), 23 were dependent of NO production (60) (Table 1). In addition, the
effect of the chemical NO donor S-nitroglutathione on
protein expression was addressed, demonstrating altered
expression levels of 19 islet protein spots, which were not
MS identified (60). This suggests that the majority of
protein changes observed in response to IL-1 are independent of NO production. It is not clear whether the plethora
of changes observed might be secondary to the early
effects of just one or very few proteins (e.g., one involved
in mitochondrial energy generation). In addition, the cellular specificity of the observed changes in islet protein
expression levels remains to be elucidated. However, most
of the changes seen in the rat islets are reproduced in
DIABETES, VOL. 50, MAY 2001
cytokine-exposed RIN-cells (data not shown). Our findings
may offer relevant mechanisms for ␤-cell death after cytokine exposure.
Whether IL-1–induced changes in protein expression
levels in rat islets in vitro will also reflect pathogenically
important changes in ␤-cells in rats spontaneously developing type 1 diabetes remains to be determined. Preliminary
observations using 2D gel studies of excised syngeneic
islet transplants from different time points posttransplantation in BB-DP rats (61) suggest that the approach may be
useful for studies of type 1 diabetes pathogenesis in vivo.
Interestingly, the exquisite ␤-cell sensitivity to cytokine
toxicity may be an acquired trait developed during ␤-cell
maturation (62).
The data presented should be interpreted with some
caution. As described, the level of expression of 105 rat
islet protein spots was consistently and significantly changed
after IL-1 exposure. Only these proteins have been analyzed further. In vitro 58% of these have been positively
identified so far, and the cellular specificity and significance of the changes remain to be elucidated. Identification of the remaining 42% should thus consolidate the
dynamic picture that is emerging. Surprisingly, some proteins previously shown to be modified by IL-1 (e.g., iNOS,
insulin, GAD, and MnSOD) were not identified in the
present experiments. It should be noted that protein expression changes that did not meet strict statistical criteria
defined in our previous article (25) might be important.
IL-1 effects, which result in posttranslational modifications
(e.g., a phosphorylation without an overall change in expression level of that protein) were picked up in the
present study. Some of the changes detected could also be
due to changes in the rate of protein turnover. Furthermore, it should be noted that the protein expression
changes were studied using only one IL-1␤ concentration
and one set of experimental conditions. Using various
concentrations of IL-1␤ or combinations of cytokines as
well as incubation and labeling periods of different length
may well produce additional results describing an even
more complicated and dynamic picture. Nevertheless, the
data presented represent the hitherto most detailed and
complex picture of the molecular processes leading to
␤-cell destruction in vitro. Although the picture is complicated and far from complete, we are looking at the ailing
␤-cell through a new window, and the challenge now is to
learn to fully understand what we see.
ACKNOWLEDGMENTS
This study was in part supported by the Juvenile Diabetes Foundation International (grant no. DK-96-012), the
Danish Diabetes Association, the Danish Medical Research Council (grant no. 9502027), and the Danish National Science Research Council (grant no. 9601730).
The skillful technical assistance of Susanne Munch,
Rikke Bonne, Lene A. Jakobsen, Andrea Lorentzen, Lotte
Christensen, and Viola Mose Larsen is highly appreciated.
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