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INTRODUCTION |
Insulin resistance in skeletal muscle, defined as reduced
insulin-stimulated glucose disposal, is a characteristic feature of
type 2 diabetes mellitus
(T2DM)1 and is believed to be
largely accounted for by reduced non-oxidative glucose metabolism
(1-3). Furthermore, insulin stimulation of glucose oxidation and
suppression of lipid oxidation is significantly impaired in patients
with T2DM (2, 3). Conversely, in the basal, fasting state increased
glucose oxidation and reduced lipid oxidation is seen in skeletal
muscle of insulin resistant subjects, whether caused by T2DM or obesity
alone (4). These defects suggest an impaired capacity to switch between
carbohydrate and fat as oxidative energy sources in insulin-resistant
subjects. Together with reports of reduced oxidative enzyme activity
and dysfunction of mitochondria in skeletal muscle of patients with T2DM (4-6) and the fact that mitochondrial DNA defects cause T2DM
through impairment of oxidative phosphorylation (7, 8), these
abnormalities in fuel metabolism have led to the hypotheses that
perturbations in skeletal muscle mitochondrial metabolism (6, 9, 10)
and defects in the signaling pathways of AMP-activated protein kinase
(AMPK) are implicated in the pathogenesis of T2DM (11). That rates of
fuel oxidation and mitochondrial function can affect glucose uptake and
glycogen synthesis has been reported earlier (12, 13). In
addition, both chronic activation of AMPK and induced expression of the
transcriptional co-activator of peroxisome proliferator-activated
receptor
, PGC-1, result in improved mitochondrial biogenesis
concomitant with increases in GLUT4 protein content in skeletal muscle
(14-16). The reduction in oxidative enzyme capacity in patients with
T2DM could be attributed to an increased proportion of glycolytic, type
2 muscle fibers (17). However, reduced oxidative enzyme capacity
seems to be equally present in all muscle fiber types in patients with
T2DM (5).
An increasing number of enzymes and metabolic pathways has been
suggested to be involved in the development of skeletal muscle insulin
resistance in T2DM. Therefore, there is a growing demand for techniques
able to evaluate proteins from many signaling and metabolic pathways
simultaneously. The recently introduced technique of proteome analysis,
high resolution two-dimensional (2-D) gel electrophoresis followed by
protein identification using mass spectrometry (MS) and data base
searching, offers the possibility to study a large number of proteins
and their post-translational modifications simultaneously (18).
In the past, primarily defects in the insulin-stimulated state have
been studied. However, as discussed above, there are several reasons to
look at T2DM as a phenotype described by poor adaptations to fasting
conditions, as well (4). To identify protein markers of T2DM in the
fasting state, we compared the protein expression profile in skeletal
muscle from obese patients with T2DM and age- and gender-matched
healthy control subjects using the methods of quantitative proteome analysis.
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MATERIALS AND METHODS |
The study was approved by the Local Ethics Committee and was
performed in accordance with the Helsinki Declaration.
Muscle Samples--
Muscle samples were obtained from 10 patients with T2DM and 10 healthy age- and gender-matched control
subjects. Five patients with T2DM were treated by diet alone, and five
patients were treated by low doses of either sulfonylurea or metformin.
These drugs were withdrawn 1 week prior to the study. The patients were
all GAD65 antibody-negative and without signs of diabetic retinopathy, nephropathy, neuropathy, or macrovascular complications. All subjects had normal results on screening blood tests of hepatic and renal function. The control subjects had normal glucose tolerance and no
family history of diabetes. Participants were instructed to avoid
vigorous exercise for 48 h before the study, which was carried out
after a 10-h overnight fast. Fasting blood samples were analyzed for
glucose (Glucose Analyzer II; Beckman Instruments, Fullerton, CA), free
fatty acids (FFA) (Wako Chemicals Gmbh, Neuss, Germany), insulin, and
C-peptide (Wallac Oy, Turku, Finland). Percutaneous needle biopsies
were obtained from the vastus lateralis muscle under local anesthesia
using a biopsy pistol, and the muscle specimens (~25 mg) were
immediately blotted free of blood, fat, and connective tissue and
frozen in liquid nitrogen.
Sample Preparation--
The frozen muscle samples were
homogenized for 25 min in 100 µl of ice-cold DNase/RNase buffer (20 mmol/liter Tris-HCl buffer, pH 7.5, containing 30 mmol/liter NaCl, 5 mmol/liter CaCl2, 5 mmol/liter MgCl2, and 25 µg/ml RNase A, 25 µg/ml DNase I (Worthington, Freehold, NJ)). After
homogenization, the samples were lyophilized overnight and then
dissolved in 120 µl of lysis buffer (7 M urea (ICN
Biomedicals), 2 M thiourea (Fluka), 2% CHAPS (Sigma),
0.4% dithiothreitol (Sigma), 0.5% Pharmalyte 3-10, and 0.5%
Pharmalyte 6-11 (Amersham Biosciences) by shaking overnight. The
homogenization was carried out in a buffer without kinase and
phosphatase inhibitors (salts) to avoid destruction of the first
dimensional gels. As this step was carried out at 0-4 °C, we
assumed the activity of potential kinases and phosphatases to be very
low and that the phosphorylation state of most proteins was stable. To
confirm this, we have, after the present study, performed similar 2-D
gels, in which the muscle specimens were solubilized directly in the
lysis buffer for running the first dimensional gel. The pattern of the
proteins on these 2-D gels and in particular the relative abundance of
the different phosphoisoforms identified by MS showed no difference
from the 2-D gels presented in this study.
Protein Determination--
The protein concentration in the
muscle samples was determined using the Bradford method, which was
adopted for use with lysis buffer as described before (19).
2-D Electrophoresis--
First dimension gel electrophoresis was
performed on IPG covering the pH range from 4 to 7 (Amersham
Biosciences). Rehydration buffer for IPG 4-7 strips was identical with
lysis buffer used for sample preparation, and the sample was applied by
in-gel rehydration. 400 µg of protein were loaded on each gel.
Focusing was performed on a Multiphor II at 20 °C using a
voltage/time profile linearly increasing from 0 to 600 V for 2.25 h, from 600 to 3500 V for 1 h, and 3500 V for 13.5 h. After
focusing, strips were equilibrated twice, each for 15 min in
equilibration buffer (6 M urea, 2% SDS, 30% glycerol, 50 mM Tris-HCl, pH 8.8, 1% dithiothreitol). Gels were frozen
at
80 °C between the equilibration steps. SDS-PAGE second
dimension was performed using the ProteanTM II Multi Cell
2-D electrophoresis system (Bio-Rad) and laboratory-made single
percentage gels (12.5% acrylamide;
acrylamide:N,N'-ethylene-bis-acrylamide ratio was
200:1). The gels were run overnight at 20 °C at constant current.
Running buffer was recirculated to maintain pH, SDS, temperature, and salt concentrations.
Protein Visualization and Computer Analysis--
After the
second dimension, skeletal muscle proteins were visualized using a
silver-staining method as described (19). All gel images were analyzed
by the same person using a Bio Image computer program (version 6.1; Bob
Luton, Ann Arbor, MI). For comparison we have used 2-D gels from six
subjects in the control group and nine subjects in the diabetes
group (Table I). The remaining
gels revealed that some protein degradation had occurred, and therefore
these gels were excluded from the analysis. The expression of each
protein was measured and expressed as its percentage integrated optical
density (%IOD) (a percentage of the sum of all the pixel gray level
values on and within the boundary of the spot in question compared with
that of all detected spots). Images from each group were then matched,
edited, and compared statistically. The average value of spot %IOD and
S.D. were calculated for each protein in each group and then compared
using the two-sided Student's t test. Protein spots whose
expression was found different between the two groups at the
significance level of p < 0.05 were selected for
further analysis. For correlation analysis Spearman's rho was
used.
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Table I
Fasting characteristics of study subjects
Clinical characteristics of subjects whose 2-D gels were included in
the computerized image analysis. Data represent means ± S.E. NS,
not significant.
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Mass Spectrometry and Protein Identification--
Proteins of
interest were cut out from the gels and, after in-gel digestion,
analyzed by mass spectrometry using a Bruker REFLEX matrix-assisted
laser desorption/ionization time-of-flight (MALDI-TOF) mass
spectrometer (19, 20). The mass spectra obtained were internally
calibrated using trypsin autodigestion peptides, and the masses were
used to search the NCBI data base using the ProFound, FindPept, and
FindMod programs (www.proteometrics.com). Data base searches
were performed using the following attributes with minor modification
needed for each program: all species, no restrictions for molecular
weight and protein pI, trypsin digest, one missed cleavage allowed,
cysteines modified by acrylamide, and oxidation of methionines
possible, mass tolerance between 0.1 and 0.5 Da. Identification was
considered positive when at least five peptides matched the protein
with no sequence overlap.
[32P]Labeling of Human Myoblasts--
Human
skeletal muscle cell cultures were established as described previously
(21). Myoblasts were grown in 12-wells plates, and growing cell medium
was changed to Dulbecco's modified Eagle's medium containing 5 mM glucose and supplemented with 10% fetal calf serum 1 day before the labeling experiment. Prior to labeling, cells were
incubated in serum-free Dulbecco's modified Eagle's medium containing
0.2% bovine serum albumin for 2.5 h. Phosphate groups in proteins
of human myoblasts were labeled biosynthetically by incubating them in
300 µl of serum-free phosphate-free Dulbecco's modified Eagle's
medium (ICN Biomedicals) supplemented with 2 mM
L-glutamine (Invitrogen), 0.2% bovine serum
albumin, and 300 µCi of [32P]orthophosphate (Amersham
Biosciences) for 2.5 h. Immediately after, labeling medium was
removed, and cells were lysed in 400 µl of lysis buffer as described
above. Determination of [32P]orthophosphate incorporation
into myoblast proteins was performed using trichloroacetic acid
precipitation as described (19). 2-D gel electrophoresis was run as
described above loading a cell lysate volume corresponding to 4 × 105 cpm on the gel. [32P]labeled proteins of
myoblasts were visualized by exposing dried gels to phosphorimaging
plates (AGFA).
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RESULTS |
Quantitative Proteome Analysis--
Using computerized image
analysis 489 spots in each gel image were matched and quantitated.
Fifteen protein spots were expressed at statistically significant
different levels in the two groups (Fig.
1). These potential protein markers of
T2DM were excised from the 2-D gels for identification by MALDI-TOF-MS
analysis. Eleven of these protein spots were positively identified:
three metabolic enzymes, two heat shock proteins, and different
isoforms of three structural proteins (Table
II). Of the metabolic enzymes, ATP
synthase
-subunit (ATPsyn
) and creatine kinase, brain isoform (CK-B) were significantly down-regulated, whereas phosphoglucomutase-1 (PGM-1) was significantly up-regulated in skeletal muscle of patients with T2DM. Heat shock protein (HSP) 90
and 78-kDa glucose-regulated protein (GRP78) were both significantly up-regulated in diabetic muscle. Of the structural proteins, four isoforms of
-1 chain of
type VI collagen (
1(VI) collagen) and one isoenzyme of myosin regulatory light chain 2 (MRLC2-B) were significantly up-regulated, whereas another isoenzyme of myosin regulatory light chain 2 (MRLC2-A) was significantly down-regulated in diabetic muscle.

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Fig. 1.
Protein markers of type 2 diabetes in
skeletal muscle. Representative 2-D gel image of human skeletal
muscle from a type 2 diabetic subject. Protein spots were separated in
the first dimension by IPG gels (covering the pH range from 4 to 7) and
visualized by silver staining. The numbers correspond to the
protein spots that were significantly up-regulated
(underlined) or down-regulated in skeletal muscle of type 2 diabetic subjects. Of these fifteen protein spots, eleven were
positively identified by MALDI-MS (Table II).
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Table II
Protein markers of type 2 diabetes in skeletal muscle identified by
MALDI-TOF MS analysis
Identified protein markers of type 2 diabetes in skeletal muscle are
given with their database accession numbers, theoretical molecular
weight (mW) and pI. The expression levels of these protein markers are
given as mean ± S.E. of the percentage integrated optical density
(%IOD) of proteins.
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In the diabetic group the levels of ATPsyn
(spot 180) and CK-B (spot
541) correlated negatively with fasting plasma glucose values
(r =
0.75; p < 0.05) and
(r =
0.82; p < 0.01), respectively (Fig. 2, A and B),
whereas the levels of
1(VI) collagen (spot 11) correlated positively
with fasting plasma glucose values (r = 0.67;
p < 0.05). Furthermore, the levels of ATPsyn
(spot
180) correlated positively with the levels of CK-B (spot 541)
(r = 0.71; p < 0.05) (Fig.
2C) and negatively with the levels of
1(VI) collagen (spot 11) (r =
0.72; p < 0.05) in the diabetic group. In contrast, in the control group the
levels of ATPsyn
(spot 180) correlated negatively with fasting FFA
values (r =
0.81; p < 0.05) rather
than fasting plasma glucose values (Fig. 2D), and the levels
of PGM-1 (spot 456) correlated positively with insulin
(r = 0.95; p < 0.01). No other
correlations among glucose, FFA, insulin, or C-peptide levels and the
identified potential protein markers of T2DM were observed in the two
groups. In particular, none of these protein markers correlated with
body mass index either in the entire study population or within the
study groups.

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Fig. 2.
Relation among
ATPsyn , CK-B, and plasma glucose in type 2 diabetes. Correlation of fasting plasma glucose levels with the
expression of the down-regulated ATPsyn isoform and with the
down-regulated CK-B isoform in skeletal muscle in type 2 diabetic
subjects (T2DM) (A and B).
C, correlation between the expression of the down-regulated
ATPsyn isoform and the expression of down-regulated CK-B isoform in
skeletal muscle in T2DM; D, correlation of fasting plasma
FFA levels with the expression of the down-regulated ATPsyn isoform
in control subjects.
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Identification of Protein Marker Isoforms--
CK-B, HSP 90
,
GRP78, PGM-1, and MRLC2 have been reported previously (22-26) to be
phosphorylated. Several of the identified protein spots were located
within a row of three-six protein spots with the same molecular weight
indicating multiple post-translational modifications (possibly
phosphorylation) of the same protein (Fig. 1). These protein spots were
excised and subjected to MALDI-MS analysis. Two additional isoforms of
HSP 90
, GRP78, PGM-1,
1(VI) collagen, MRLC2-A, and MRLC2-B and
three additional isoforms of ATPsyn
were positively identified (Fig.
3). Calculation of sums of expression of
the different isoforms of each protein marker showed that the sum of
expression of HSP 90
, GRP78,
1(VI) collagen, and ATPsyn
were
significantly up- or down-regulated in the same direction as the
protein markers (Table III). This was
also the case with the sum of expression of MRLC2-A and MRLC2-B,
although these differences did not reach statistical significance
(Table III). The relative abundance of the down-regulated ATPsyn
isoform (spot 180) was significantly reduced in muscle of patients with T2DM, as well (p = 0.03), indicating a role for
post-translational modification of this protein. Interestingly, in the
diabetic group the sum of expression of ATPsyn
and
1(VI) collagen
correlated significantly with fasting plasma glucose values
(r =
0.76 and r = 0.70;
p < 0.05, respectively), and the sum of expression of ATPsyn
correlated significantly with the sum of expression of
1(VI) collagen (r =
0.87; p < 0.01).

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Fig. 3.
Isoforms of protein markers of type 2 diabetes. Enlarged regions of the silver-stained 2-D gel
demonstrating the potential additional isoforms of HSP 90 , GRP78,
PGM-1, 1(VI) collagen, ATPsyn , MRLC2-A, and MRLC2-B. The
asterisks indicate the protein markers (Table II), and the
arrows indicate the additional isoforms positively
identified by MALDI-MS analyses (summed up in Table III).
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Table III
Sum of expression of protein markers isoforms
The calculated sum of expression of the protein marker isoforms (Fig.
2) given as means ± S.E. of the percentage integrated optical
density (%IOD) of proteins.
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Phosphorylation of ATP Synthase
-Subunit--
The
identification of four isoforms of ATPsyn
with identical molecular
weights but different pI values, suggested that ATPsyn
is also
phosphorylated in vivo (Fig. 3). To further characterize the
modification of ATP synthase
-subunit, we carried out 2-D gel
electrophoresis of [32P]labeled human skeletal muscle
cells (myoblasts) in culture. These 2-D gels revealed that all four
identified ATPsyn
isoforms are in fact phosphorylated isoforms (Fig.
4A) and that a putative non-modified variant was below the level of detection by silver staining. MALDI-MS analysis and data base searching for phosphopeptides from the tryptic digest of the three most basic phosphoisoforms of
ATPsyn
, including the down-regulated ATPsyn
spot (180),
demonstrated the presence of a phosphorylated residue most likely at
position Thr-213 in the nucleotide-binding domain of ATPsyn
(Fig. 4,
B and C). Tyrosine sulfation may give rise to the
same pattern on 2-D gels and the same increase in peptide mass (80 Da)
as phosphorylation. However, according to the consensus features of a
tyrosine sulfation site, such sites are not present in the sequence of
ATPsyn
. The [32P]labeling of all four ATPsyn
isoforms and the MS data therefore indicate that ATPsyn
is regulated
by multi-site phosphorylation.

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Fig. 4.
Phosphorylation of ATP synthase
-subunit in human skeletal muscle.
A, enlarged regions of 2-D images of protein spots separated
in the first dimension by IPG gels (covering the pH range from 4 to 7)
showing the four isoforms of ATPsyn . Visualization of protein spots
by [32P]labeling of cultured human skeletal muscle cells
(myoblasts) compared with silver-staining of proteins of in
vivo human skeletal muscle indicated that all of the four
identified ATPsyn isoforms are phosphorylated proteins. The
asterisk indicates the ATPsyn isoform, which was
down-regulated in muscle of type 2 diabetic subjects. The MALDI peptide
mass map of the most abundant ATPsyn isoform showed the presence of
a phosphorylated peptide (two peaks with a mass difference
of 80 Da) (B). The sequence coverage of the most abundant
ATPsyn isoform was 65%, and 29 of 45 measured peptides were matched
to this protein as indicated (bold) (C). The
potential phosphorylation site of the phosphorylated peptide (marked by
a box) was Thr-213 within the nucleotide-binding region
(underlined).
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DISCUSSION |
Using the methods of proteome analysis we have
identified and characterized eight potential protein markers of T2DM in
skeletal muscle in the fasting state. Although proteome analysis is
somewhat limited by narrow dynamic ranges of silver staining and
difficulties in MS identification of low abundant proteins recovered
from fixed and or stained gels, it is the only technique that provides
the possibility to quantitatively study global changes in expression profile of proteins, as well as certain post-translational protein modifications in a given cell or tissue. Because of the method of
sample preparation used in this study the interpretation of the
relative abundance of the specific protein marker phosphoisoforms should be done with some caution. However, in the present study MS
identification of ~75% of the protein markers and several additional isoforms demonstrates that combined 2-D gel and MS technology can be
powerful tools for the study of molecular processes underlying a
complex disease such as T2DM.
Our findings indicate that the catalytic
-subunit of
F1-ATP synthase is regulated by phosphorylation and that
the expression and phosphorylation of ATPsyn
might be altered in
skeletal muscle of patients with T2DM. Because there is no report to
date on the phosphorylation of human ATPsyn
it is uncertain how such
post-translational modification interacts with the proposed
binding-change model for ATP synthesis, in which conformational changes
in the nucleotide-binding sites of the three
-subunits are coupled
to the catalytic activity of F1-ATP synthase (27). That
phosphorylation of human ATPsyn
may play a role for the catalytic
activity of F1-ATP synthase is suggested by the recent
observation that phosphoserine/phosphothreonine-binding 14-3-3
proteins were found to be associated with mitochondrial ATP synthase in
plants in a phosphorylation-dependent manner through direct
interaction with the
-subunit of F1-ATP synthase and
that the activity of ATP synthase was reduced by recombinant 14-3-3 protein (28). Consistent with the reduced expression of ATPsyn
in
our study, several other studies argue for a lower ATP synthase activity in diabetic muscle. Thus, gene expression of several subunits
from the other four complexes of the mitochondrial electron transport
chain were found to be decreased in skeletal muscle of
streptozotocin-diabetic mice (29), and during fasting conditions reduced activity of cytochrome c oxidase and citrate
synthase activity, as well as reduced overall activity of the
respiratory chain, have been demonstrated in skeletal muscle of
patients with T2DM (4-6). Moreover, mutations in mtDNA can, via
impaired oxidative phosphorylation, cause T2DM, with both muscle
insulin resistance and impaired insulin secretion (7-8). Indeed,
decreased ATP synthase activity and increased formation of reactive
oxygen species (ROS) have been reported in hybrids constructed from
mitochondria of patients with T2DM (9). However, whether the
down-regulation of a specific ATPsyn
phosphoisoform is a mechanism
to maintain normal activity despite reduced total levels of ATPsyn
or whether it reflects an altered activity of ATPsyn
per
se warrants further studies. Endurance training up-regulates
ATPsyn
protein levels in soleus muscle of rats (30). Thus, a
sedentary lifestyle and reduced amounts of oxidative type 1 fibers in
type 2 diabetic subjects (17) might play a role in the altered levels
of ATPsyn
observed in our study. In control subjects we observed a
negative correlation between the down-regulated ATPsyn
phosphoisoform and fasting plasma-free fatty acids. In contrast, in the
patients with T2DM both this phosphoisoform and the total expression of ATPsyn
correlated inversely with fasting plasma glucose. These findings are consistent with a predominant reliance on lipid oxidation in the fasting state in muscle of lean non-diabetic subjects, which is
changed to a poor reliance on lipid oxidation in muscle of patients
with T2DM (4, 5). Recently, mRNA levels of the ATP synthase
-subunit, as well as of GLUT-4, were shown to be decreased 2-3-fold
in skeletal muscle from patients with T2DM, in whom insulin treatment
was withdrawn for 14 days (31), indicating that altered expression of
ATPsyn
could be secondary either to hyperglycemia or to relative
deprivation of the effects of insulin signaling (insulin resistance).
Mitochondria are the intracellular sites for fuel oxidation and ATP
production, and that mitochondrial dysfunction might play a key role
for the impaired glucose metabolism observed in muscle of patients with
T2DM is supported by the predominantly maternal transmission of T2DM in
Caucasian populations (32) and by several recent studies demonstrating
that regulation of mitochondrial function and glucose uptake seem to be
tightly coupled; thus, both chronic activation of AMPK (14) and induced
expression of PGC-1 (15, 16) result in improved mitochondrial
biogenesis concomitant with increases in GLUT4 protein.
PGM-1 is a glycolytic enzyme that plays a pivotal role in glycogen
metabolism, catalyzing the interconversion of glucose-1-phosphate and
glucose-6-phosphate. The activity of PGM-1 is increased by phosphorylation and is reduced in type 1 fibers of skeletal
muscle and with endurance training (23, 33). The increased levels of
the most acidic (phosphorylated) isoform observed in this study suggest
that PGM-1 activity could be increased in skeletal muscle of patients
with T2DM consistent with a reduced amount of type 1 fibers (17) and an
increased glycolytic-to-oxidative ratio in the fasting state (4), as
well as a sedentary lifestyle in such subjects. Together with the
absence of correlation between the levels of PGM-1 and insulin in
patients with T2DM, these observations raise the question of whether an
increase in PGM-1 may play a role in the impaired glucose metabolism
observed in patients with T2DM (1-3).
CK-B levels in skeletal muscle are much lower than in cardiac muscle
(34) but are higher in type 1 muscle fibers and have been reported to
increase with endurance training and to be positively correlated with
oxidative enzyme capacity irrespective of muscle fiber type (35). As
with ATPsyn
and PGM-1, the reduced levels of a CK-B isoform in
skeletal muscle of patients with T2DM seen here could be explained by
reduced amounts of type 1 fibers (17), reduced oxidative enzyme
capacity (4, 5), and a sedentary lifestyle in such patients. In
skeletal muscle of streptozotocin-diabetic mice a reduction in mRNA
levels of CK-B was observed, together with decreased mRNA levels
of several subunits in the electron transport chain (29). These data
suggest a specific role for CK-B in mitochondrial fuel oxidation. We
observed a negative correlation between CK-B and plasma glucose levels
and a positive correlation between CK-B and the down-regulated
ATPsyn
phosphoisoform in patients with T2DM, which may indicate that
down-regulation of this ATPsyn
phosphoisoform actually reduces the
catalytic activity of F1-ATP synthase. Allowing resynthesis
of ATP from phosphocreatine, creatine kinase plays a key role in the
energy metabolism of heart and skeletal muscle, and the observed
correlations support the hypothesis of a functional coupling of
creatine kinase isoenzymes to mitochondrial energy production and
glycolysis (36). Interestingly, the creatine kinase system is regulated
by AMPK via phosphocreatine/creatine and ATP/AMP ratios (36, 37), and
perturbations in the AMPK signaling system were suggested recently (11)
as potential key players in the development of T2DM.
GRP78 and HSP 90 are stress-inducible proteins belonging to the group
of heat shock proteins and function as molecular chaperones. The
in vivo functions of both proteins are dependent on an
inherent ATPase activity and phosphorylation (24, 25, 38, 39). Conditions giving rise to increased levels and activity of GRP78 are
followed by a decreased phosphorylation of GRP78 (25), whereas phosphorylation of HSP 90 seems to be linked to its chaperoning function (24). We observed increased levels of GRP78 in patients with
T2DM, and of the three isoforms identified only the most basic
(non-phosphorylated) and most active isoform was significantly up-regulated. The increase in HSP 90
levels in patients with T2DM
was caused mainly by an increase in an acidic (phosphorylated) isoform.
What type of cellular stress induces HSP 90
and GRP78 in muscle of
patients with T2DM is currently unknown. Both sustained hypoglycemia
and hyperglycemia (40), as well as oxidative stress, lead to increases
in GRP78 expression (41). Hyperglycaemia increases ROS formation in
cultured endothelial cells (42), and increased ROS formation and
oxidative stress in heart muscle leads to increased mRNA levels of
HSP 70 and HSP 90 (43). However, we observed no relationship between
fasting plasma glucose levels and the levels of HSP 90
or GRP78
arguing against hyperglycemia as the direct cause of cellular stress.
As mentioned above increased ROS formation and oxidative stress caused
by mitochondrial dysfunction have been shown in hybrids constructed
from mitochondria of patients with T2DM (9). Increased ROS formation
and oxidative stress lead to increased amounts of incorrectly folded
and denatured proteins, which induces the expression of cellular
chaperones such as GRP78 and HSP 90
(44). Interestingly, both GRP78
and HSP 90 have been shown to play a role in the
post-translational processing of certain mutant insulin receptors
linking these molecular chaperones to severe insulin resistance (45,
46). In addition, HSP 90
has been shown to play an important role in
maintaining the activity of protein kinase B (47), which seems to be
involved in the regulation of insulin-mediated glucose transport and
glycogen synthesis, two processes shown to be impaired in T2DM (1). Consistent with our data, mRNA expression of a heat shock protein (HSP 70) was found to be increased in skeletal muscle of patients with
T2DM both before and after withdrawal of insulin treatment (31).
Further studies are needed to establish what specific roles increased
levels of certain stress proteins such as HSP 90
, GRP78, and HSP 70 play in the pathogenesis of insulin resistance in muscle of patients
with T2DM.
The function of both collagen type VI and MRLC2 in skeletal muscle is
only partially understood, and the potential implications of the
observed changes in expression of these structural proteins are
therefore unclear. However, mutations in the MRLC2 gene have been shown
to cause cardiac and skeletal muscle myopathy with ragged red fiber
histology similar to mitochondrial myopathy (48). This relationship
between MRLC2 mutations and their energy source (mitochondria) suggests that the observed changes might be secondary to
perturbations in skeletal muscle mitochondrial metabolism. In
vitro, hyperglycemia up-regulates type VI collagen, and
up-regulation of this collagen has been demonstrated in type 2 diabetes
in myocardium and nerves (49, 50). The observed association of type VI
collagen levels to both plasma glucose and ATPsyn
levels in diabetic
muscle suggests that increased (oxidative) stress, whether caused by hyperglycemia or mitochondrial dysfunction (9), could be responsible for the accumulation of modified long-lived proteins such as type VI collagen.
In summary, using proteome analysis we have identified eight potential
protein markers of T2DM in skeletal muscle in the fasting state. The
observed changes in expression of these protein markers could all be
linked to increased cellular stress and perturbations in skeletal
muscle mitochondrial metabolism. Two proteins important for ATP
synthesis, CK-B and ATPsyn
, were reduced in diabetic muscle and
correlated inversely with plasma glucose levels. However, whether these
changes are secondary to hyperglycemia, hyperinsulinemia, an altered
fiber type composition in muscle, or whether they are indicators of a
more primary defect in T2DM remains to be elucidated. Phosphorylation
appears to play a key, potentially coordinating role for
most of the proteins identified in this study. In particular, we
demonstrated that the catalytic
-subunit of F1-ATP
synthase is phosphorylated in vivo, which may contribute
substantially to the understanding of the regulation of ATP synthase.
These data suggest a role for phosphorylation of ATPsyn
in the
regulation of ATP synthesis and indicate that alterations in the
regulation of ATP synthesis and cellular stress proteins might
contribute to the pathogenesis of T2DM.