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18 DIABETES, VOL. 50, JANUARY 2001
Rapid Publication
Effect of Oral Creatine Supplementation on Human
Muscle GLUT4 Protein Content After Immobilization
B. Op ’t Eijnde, B. Ursø, E.A. Richter, P.L. Greenhaff, and P. Hespel
The purpose of this study was to investigate the effect
of oral creatine supplementation on muscle GLUT4
protein content and total creatine and glycogen con-
tent during muscle disuse and subsequent training. A
double-blind placebo-controlled trial was performed
with 22 young healthy volunteers. The right leg of
each subject was immobilized using a cast for 2 weeks,
after which subjects participated in a 10-week heavy
resistance training program involving the knee-exten-
sor muscles (three sessions per week). Half of the
subjects received creatine monohydrate supplements
(20 g daily during the immobilization period and 15 and
5 g daily during the first 3 and the last 7 weeks of
rehabilitation training, respectively), whereas the
other 11 subjects ingested placebo (maltodextrine).
Muscle GLUT4 protein content and glycogen and total
creatine concentrations were assayed in needle biopsy
samples from the vastus lateralis muscle before and
after immobilization and after 3 and 10 weeks of
training. Immobilization decreased GLUT4 in the
placebo group (–20%, P < 0.05), but not in the creatine
group (+9% NS). Glycogen and total creatine were
unchanged in both groups during the immobilization
period. In the placebo group, during training, GLUT4
was normalized, and glycogen and total creatine were
stable. Conversely, in the creatine group, GLUT4
increased by ~40% (P < 0.05) during rehabilitation.
Muscle glycogen and total creatine levels were higher
in the creatine group after 3 weeks of rehabilitation
(P < 0.05), but not after 10 weeks of rehabilitation. We
concluded that 1) oral creatine supplementation off-
sets the decline in muscle GLUT4 protein content that
occurs during immobilization, and 2) oral creatine
supplementation increases GLUT4 protein content
during subsequent rehabilitation training in healthy
subjects. Diabetes 50:18–23, 2001
I
t is well established that high-dose (20–25 g per day) oral
creatine intake can rapidly (3–5 days) raise muscle total
creatine content. This elevation in muscle creatine
storage is associated with increased muscle power out-
put during short high-intensity exercise. In addition, it has
been shown that long-term creatine intake can enhance the
effects of weight training on muscle volume and strength (1,2).
The use of creatine as an ergogenic supplement in sports has
prompted interest in the potential of oral creatine supplemen-
tation to treat muscle atrophy and neuromuscular diseases.
Thus, the recovery of muscle disuse atrophy due to immobi-
lization was significantly enhanced by creatine supplemen-
tation (P.H., B.O.E., M. Van Leemputte, B.U., P.L.G., V. Labarque,
S. Dymarkowski, P. Van Hecke, E.A.R, unpublished observa-
tions). Furthermore, creatine supplementation was found to
have a beneficial impact on muscle functional capacity in various
modes of mitochondrial cytopathies (3) and muscle dystrophies
(4). At the same time, evidence is accumulating to suggest that
creatine supplementation may be an effective neuroprotective
agent to treat neurodegenerative diseases (5–7).
Interestingly, a number of recent observations also indicate
that creatine supplementation might have a beneficial impact
on glucoregulation. For instance, it has been shown that the
ingestion of creatine in combination with carbohydrate
supplements can stimulate postexercise muscle glycogen
resynthesis (8), which is conceivably due to enhanced insulin-
mediated muscle glucose uptake (9). Similarly, creatine intake
in conjunction with carbohydrates was found to result in
greater muscle creatine accumulation than creatine intake
alone (10), which may be due to the fact that both glucose trans-
port and creatine transport (11) in muscle cells are stimulated
by insulin. On the other hand, a number of in vitro studies
have found that high extracellular concentrations of guanidine
compounds, including creatine, stimulate pancreatic insulin
secretion (12,13). However, the extracellular creatine con-
centrations obtained by oral creatine intake in humans do not
affect insulin secretion (14,15). Perhaps the most striking evi-
dence to suggest that creatine supplementation might be
an effective strategy to treat insulin resistance comes from a
recent study on transgenic Huntington mice. The addition of
creatine to the diet of the Huntington mice resulted in a marked
neuroprotective effect and significantly reduced the hyper-
glycemia typical of these mice, while improving the glucose
response to intravenous glucose injection (5).
Based on the above evidence, we speculate that creatine
supplementation may enhance insulin-mediated muscle
glucose uptake and glycogen synthesis, thereby beneficially
impacting whole-body glucose homeostasis. This creatine
From the Faculty of Physical Education and Physiotherapy (B.O.E., P.H.),
Exercise Physiology and Biomechanics Laboratory, Katholieke Universiteit
Leuven, Leuven, Belgium; the Department of Human Physiology (B.U., E.A.R.),
Copenhagen Muscle Research Center, University of Copenhagen, Copenhagen,
Denmark; and the School of Biomedical Sciences (P.L.G.), Queens Medical
Center, University of Nottingham, Nottingham, U.K.
Address correspondence and reprint requests to Peter Hespel, PhD,
Faculty of Physical Education and Physiotherapy, Exercise Physiology and
Biomechanics Laboratory, Tervuursevest 101, B-3001 Leuven, Belgium. E-mail:
peter.hespel@flok.kuleuven.ac.be.
Received for publication 13 September 2000 and accepted 24 October 2000.
Posted on the World Wide Web at www.diabetes.org/diabetes on 30 November 2000.
AMPK, AMP-activated protein kinase; CK, creatine kinase; DW, dry
weight; MAPK, mitogen-activated protein kinasE; RM, repetition maximum.
DIABETES, VOL. 50, JANUARY 2001 19
B. OP ‘T EIJNDE AND ASSOCIATES
response might be particularly relevant to the prevention and/or
treatment of disease states characterized by peripheral insulin
resistance, such as type 2 diabetes, obesity, and inactivity
(16). Furthermore, it is well established that muscle inactiv-
ity and training are effective stimuli to down- and upregu-
late muscle GLUT4 content and peripheral insulin sensitivity,
respectively (17). Therefore, we investigated the effect of cre-
atine supplementation on muscle GLUT4 protein content and
total creatine and glycogen concentration in healthy volunteers
during 2 weeks of leg immobilization and during 10 weeks of
subsequent rehabilitation training. This report is part of a
larger study (P.H., B.O.E., M. Van Leemputte, B.U., P.L.G., V.
Labarque, S. Dymarkowski, P. Van Hecke, E.A.R) that investi-
gated the effects of creatine supplementation on muscle func-
tional capacity during disuse atrophy in healthy subjects.
RESEARCH DESIGN AND METHODS
Subjects. A total of 13 men and 9 women, aged 20–23 years, gave their informed
written consent to participate in the study. They were instructed to abstain from
taking any medication and to avoid making any changes in their usual physi-
cal activity level and other living habits during the period of the study. How-
ever, three of the women were taking oral contraceptives for the duration of
the study. The local ethics committee approved the study protocol.
Study protocol. A double-blind study was performed over a 12-week period.
At the start of the study, subjects were systematically assigned to either a cre-
atine or a placebo group based on quadriceps muscle cross-sectional area and
maximal isometric knee-extension torque to obtain two groups of similar dis-
tribution (P.H., B.O.E., M. Van Leemputte, B.U., P.L.G., V. Labarque, S. Dymarkowski,
P. Van Hecke, E.A.R, unpublished observations). After baseline measurements had
been taken, a light polyester cast, extending from groin to ankle, immobilized
each subjects’ right leg at a knee angle of ~160° for 2 weeks. Thereafter, the
cast was removed, and the subjects underwent a standardized 10-week reha-
bilitation program. Each training session consisted of four series of 12 uni-
lateral knee-extensions on a knee-extension apparatus, at a workload of 60%
of maximal isometric knee-extension torque and at a rate of three sessions per
week. Maximal knee-extension torque was measured at a 90° knee-angle at the
start of each session using a calibrated force transducer. During the final 7
weeks of the training period, the series of four unilateral knee-extensions was
increased to six. All training sessions were supervised by one of the investi-
gators. During immobilization, the creatine group received 5 g creatine mono-
hydrate four times per day, whereas the placebo group received placebo sup-
plements (5 g maltodextrine, four times per day). During the training period,
creatine and placebo supplementation was reduced to 5 g three times per day
from week 1 to 3 and then to a single 5-g daily dose from week 4 to 10. The
creatine supplements were flavored by the addition of citrate (60 mg/g crea-
tine) and maltodextrine (940 mg/g creatine), whereas the placebo group
ingested maltodextrine containing citrate (40 mg/g maltodextrine). Creatine
and placebo powders were identical in taste and appearance. Before and
after 2 weeks of immobilization, and after 3 and 10 weeks of rehabilitation, a
percutaneous needle biopsy from the vastus lateralis muscle was
obtained. The last training session preceded muscle sampling by at least 48 h.
In addition, the subjects received a standardized dinner (855 kcal, 47% car-
bohydrate, 25% fat, and 28% protein) the evening before and a standardized
breakfast (320 kcal, 65% carbohydrate, 15% fat, and 20% protein) the morning
of muscle sampling. To collect each muscle biopsy, an incision was made
through the skin and muscle fascia under local anesthesia (2–3 ml 1% lido-
caine). During sessions 2–4, the incision was made either proximal or distal
to the incision made at an earlier session. On removal from the limb, a piece
of each muscle biopsy was immediately blotted and cleaned from visible con-
nective tissue, rapidly frozen in liquid nitrogen, and stored at –80°C for sub-
sequent biochemical and immunochemical analyses.
Biochemical and immunochemical analyses. The biopsy samples were
first freeze-dried, then washed twice in petroleum ether to remove fat, and
finally dissected free of the remaining visible blood and connective tissue. A
fraction of each sample was pulverized, and the powdered extracts were
used for spectrophotometric determination of glycogen and free creatine and
phosphocreatine concentrations (18). Another fraction was used for GLUT4
determination. An aliquot of the freeze-dried muscle was homogenized (Poly-
tron) for 30 s on ice in a buffer with the following composition: 150 mmol/l
NaCl, 1% NP
4
O, 0.5% deoxycholate, 0.1% SDS, and 50 mmol/l Tris, pH 8. The
homogenate was incubated on ice for 1 h and spun for 15 min at 13,000g, and
the supernatant (extract) was collected for analysis. Then, 100 µg of the
extract were resolved by SDS-PAGE before electroblotting to polyvinylidine
fluoride membranes. GLUT4 proteins were then detected by incubation in Tris-
buffered saline with Tween (150 mmol/l NaCl, 50 mmol/l Tris, and 0.1% Tween
20) after blocking in 1% bovine serum albumin with a specific goat polyclonal
antibody against the 13 COOH-terminal amino acids of GLUT4. Finally, GLUT4
was visualized by an alkaline phosphatase-labeled antibody and quantified on
a phosphoimager (STORM; Molecular Dynamics, Sunnyvale, CA).
Data analysis. Data are means ± SE. Muscle total creatine concentration was
calculated as the sum of free creatine and phosphocreatine. Treatment effects
(creatine versus placebo) were evaluated by a two-way analysis of variance,
which was covariate adjusted for the baseline values (Statistica; Statsoft,
Tulsa, OK). In addition to these primary analyses, we did a one-way analysis
of variance to compare the values after immobilization and rehabilitation
with the corresponding baseline values within each group. The statistical
analyses of the GLUT4 data were performed on the raw data (densitometric
counts). P < 0.05 was considered statistically significant.
RESULTS
Muscle GLUT4 content. Muscle GLUT4 concentrations were
expressed relative to the corresponding baseline values that
were set equal to 1 (Fig. 1). Muscle GLUT4 content at baseline
was similar between the groups. In the placebo group, 2 weeks
of immobilization decreased GLUT4 content on an average of
22% (range –10 to –35%, P < 0.05). Conversely, in the creatine
group, muscle GLUT4 protein was stable (+9% NS). In the
placebo group, the rehabilitation training restored muscle
GLUT4 content within 3 weeks to the baseline value, where
it remained. However, in the creatine group, muscle GLUT4
content progressively increased during the 10-week rehabili-
tation period to a value that was ~40% higher than in the
placebo group at the end of the study (P < 0.05).
Muscle glycogen. The initial muscle glycogen concentration
was 407 ± 43 mmol/kg dry weight (DW) in the placebo group
versus 379 ± 19 mmol/kg DW in the creatine group (NS)
(Fig. 2). Immobilization did not change muscle glycogen con-
centration in either group. However, during the initial 3
weeks of rehabilitation training, muscle glycogen markedly
increased in the creatine group (P < 0.05), whereas it did not
significantly change in the placebo group. Thus, after 3
weeks, muscle glycogen concentration was higher (P < 0.05)
in the creatine group (660 ± 70 mmol/kg DW) than in the
placebo group (520 ± 60 mmol/kg DW). However, during the
final 7 weeks of rehabilitation training, muscle glycogen
reverted to baseline values in both groups.
Muscle creatine content. The muscle phosphocreatine
and free creatine concentrations at baseline were similar
between both groups (Table 1). During immobilization,
phosphocreatine concentration decreased to ~15% below the
baseline value in the placebo group (P < 0.05). This decrease
was negated by creatine supplementation (P < 0.05). In
the placebo group, muscle phosphocreatine concentration
returned to the preimmobilization baseline level within the
initial 3 weeks of the rehabilitation period, after which it
remained stable. On the other hand, in the creatine group,
compared with the placebo group, the muscle phosphocrea-
tine concentration increased to ~12% above baseline value
after 3 weeks of rehabilitation (P < 0.05). However, this
increase above baseline in phosphocreatine was reversed
during the final stage of the rehabilitation period. Throughout
the study, the muscle free creatine concentrations were not
significantly different between the placebo and the creatine
groups. In the placebo group, muscle total creatine concen-
tration was not significantly changed compared with the base-
line value during either immobilization or rehabilitation. Yet
20 DIABETES, VOL. 50, JANUARY 2001
CREATINE INTAKE AND MUSCLE GLUT4
in the creatine group, compared with the placebo group, the
muscle total creatine concentration was higher at the end of
the immobilization period, as well as after 3 weeks of reha-
bilitation (P < 0.05). However, along with the declining mus-
cle phosphocreatine levels, muscle total creatine returned to
baseline by the end of the study.
DISCUSSION
Our study investigated the impact of creatine supplementa-
tion on muscle GLUT4 content and glycogen and total crea-
tine concentrations in healthy subjects during 2 weeks of
voluntary leg immobilization followed by 10 weeks of reha-
bilitation training. Our data are the first to show that creatine
supplementation prevents the loss of GLUT4 protein during
muscle disuse and increases muscle GLUT4 content above
normal levels during subsequent rehabilitation. Furthermore,
muscle glycogen concentration was increased during the ini-
tial stages of the creatine supplementation.
Glucose transport across the plasma membrane is the rate-
limiting step for glucose metabolism. Hence, muscle GLUT4
content is a primary determinant of insulin-stimulated muscle
glucose uptake and metabolism (16). Thus, increasing muscle
GLUT4 content by transgenic overexpression or by increased
contractile activity enhances maximal insulin-stimulated muscle
FIG. 1. Effect of creatine supplementation on muscle GLUT4 protein content during immobilization and subsequent rehabilitation training.
Data are means ± SE (n = 8) and are expressed relative to the baseline value that was set to be equal to 1. Muscle samples were taken from
the vastus lateralis muscle before and after 2 weeks of immobilization and after 3 and 10 weeks of rehabilitation of the right leg. During immo-
bilization and rehabilitation, subjects ingested creatine monohydrate () or placebo (). See RESEARCH DESIGN AND METHODS for further details.
*Significant treatment effect compared with placebo, P < 0.05; §significant time effect compared with the preimmobilization value.
FIG. 2. Effect of creatine supplementation on muscle glycogen concentration during immobilization and subsequent rehabilitation training.
Data are means ± SE (n = 8). Muscle samples were taken from the vastus lateralis muscle before and after 2 weeks of immobilization and after
3 and 10 weeks of rehabilitation of the right leg. During immobilization and rehabilitation, subjects ingested creatine monohydrate () or placebo
(). See RESEARCH DESIGN AND METHODS for further details. *Significant treatment effect compared with placebo, P < 0.05; §significant time effect
compared with the preimmobilization value.
DIABETES, VOL. 50, JANUARY 2001 21
B. OP ‘T EIJNDE AND ASSOCIATES
glucose uptake. Conversely, reducing the content of GLUT4
by GLUT4 knockout, denervation, or aging impairs insulin-
mediated muscle glucose uptake (19). Our data, therefore, sug-
gest that creatine supplementation in humans may increase
insulin sensitivity by increasing muscle GLUT4 content.
Over the last decade, substantial evidence has accumu-
lated to show that endurance exercise training elevates mus-
cle GLUT4 content and insulin-stimulated glucose uptake in
both healthy (17,20–28) and insulin-resistant muscles (29,30).
In this respect, the current study shows that in healthy indi-
viduals, a low volume (3 weekly sessions) of moderate resis-
tance training (60% of 1 repetition maximum [RM]), in contrast
with endurance training (23–26, 28) or daily maximal resis-
tance training (31), is not a sufficient stimulus to increase
muscle GLUT4 content. Ten weeks of rehabilitation training
per se did not increase muscle GLUT4 content above the
baseline level (Fig. 1). However, the same training regimen in
conjunction with oral creatine supplementation resulted in
a marked increase of muscle GLUT4 protein content. In fact,
our observations indicate that oral creatine supplementa-
tion can probably increase GLUT4 protein content in skele-
tal musculature independent of exercise training. In keeping
with earlier observations (17,20–22,31,32), muscle decondi-
tioning by immobilization in the placebo subjects reduced
GLUT4 protein content (~20%). Nevertheless, at the end of the
immobilization period, GLUT4 content in the creatine group
tended to increase by ~10%, which resulted in a 30% difference
in muscle GLUT4 between placebo and creatine supplemen-
tation in the absence of a training stsore, it is reasonable to con-
clude that creatine supplementation can increase GLUT4 pro-
tein content in human musculature during episodes of either
reduced or increased physical activity.
Based on the current knowledge, it is difficult to reveal the
molecular basis for the increase in muscle GLUT4 content that
occurs during creatine supplementation. It has recently been
observed in rats that short-term administration of amino-
imidazole-4-carboximide riboside, an AMP-activated protein
kinase (AMPK) agonist, increases muscle GLUT4 content
(33). Creatine administration that increases AMPK activity by
decreasing the phosphocreatine-to-creatine ratio (34) may,
thus, explain the increase in GLUT4 protein content in the
creatine group. And yet, in both groups the phosphocreatine-
to-creatine ratio decreased to the same degree during immo-
bilization and remained below the baseline value during the
subsequent rehabilitation period. Furthermore, it has recently
been shown that the creatine kinase (CK) and AMPK enzymes
colocalize in muscle cells (34). According to the prevailing
opinion, in skeletal muscle, such coupling should serve to
suppress muscle AMPK activity by maintaining high local
ATP:AMP and phosphocreatine-to-creatine ratios in condi-
tions of cellular stress, such as contractions (35). If anything,
this inhibitory action is enhanced by the increased muscle
phosphocreatine concentration established during the creatine
supplementation (Table 1). Thus, evidence for a possible cre-
atine-induced increase in AMPK activity has not been found.
Alternatively, there is substantial evidence to suggest that cel-
lular hydration status is an important factor controlling cellular
protein turnover (36), which in muscle cells, excluding the con-
tractile proteins, may involve other proteins important to
energy homeostasis, such as GLUT4. Creatine is cotransported
with Na ions across the sarcolemma, which initiates influx of
Cl
–
and water to balance electroneutrality and osmolality (11).
The resulting increase of cell volume may, in turn, act as an
anabolic proliferative signal, which involves activation of the
mitogen-activated protein kinase (MAPK) signaling cascade
that plays a pivotal role in muscle protein synthesis regulation
(37,38). It is warranted to further explore the possible role of
intracellular creatine content in modulating the concerted
actions of CK, AMPK, and MAPK in regulating GLUT4 syn-
thesis and degradation in muscle cells.
The bulk of glucose in the human body is stored as mus-
cle glycogen. The presence of a high muscle glycogen con-
centration, in general, indicates adequate insulin stimulation
of muscle glucose uptake and glycogen synthesis. Further-
more, a high muscle glycogen concentration is a prerequisite
for optimal endurance exercise performance (39). Robinson
et al. (8) have recently demonstrated that carbohydrate
intake in conjunction with creatine supplementation resulted
in greater postexercise muscle glycogen resynthesis than
carbohydrate intake alone. Accordingly, in the current study,
during the initial 3 weeks of rehabilitation training, muscle
glycogen concentration increased by ~30% in the placebo
group, whereas a threefold greater increase occurred in the
creatine group. This higher-than-average glycogen level,
TABLE 1
Effect of creatine supplementation on muscle creatine content during immobilization and subsequent rehabilitation training
Before After 3 weeks of 10 weeks of
immobilization immobilization rehabilitation rehabilitation
Free creatine (mmol/kg DW)
Placebo 31.3 ± 3.3 41.3 ± 3.6* 43.5 ± 5.4* 37.7 ± 2.9
Creatine 30.6 ± 2.9 48.5 ± 4.5* 53.9 ± 5.4* 43.4 ± 4.0
Phosphocreatine (mmol/kg DW)
Placebo 76.5 ± 1.8 64.9 ± 3.1* 73.8 ± 2.6 71.6 ± 2.2
Creatine 82.4 ± 6.2 80.2 ± 5.8† 89.7 ± 6.8† 75.1 ± 6.3
Total creatine (mmol/kg DW)
Placebo 108.8 ± 2.8 106.2 ± 5.7 117.3 ± 5.1 109.3 ± 3.4
Creatine 113.9 ± 8.4 128.7 ± 9.9*† 143.6 ± 11.6*† 118.5 ± 8.0
Data are means ± SE of eight observations and represent concentrations measured in needle biopsy samples obtained from vastus
lateralis muscle. Total creatine concentration was calculated as the sum of free creatine and phosphocreatine concentrations mea-
sured. Immobilization and rehabilitation procedures are described in
RESEARCH DESIGN AND METHODS. *Significant time-effect compared
with the preimmobilization value, P < 0.05; †significant treatment effect compared with placebo, P < 0.05.
22 DIABETES, VOL. 50, JANUARY 2001
CREATINE INTAKE AND MUSCLE GLUT4
established by creatine supplementation (>650 mmol/kg
DW) (Fig. 2), corresponds with common glycogen levels in
young healthy subjects after glycogen “supercompensation”
(39). Given that no dietary instructions were administered to
the subjects, our findings suggest that the addition of creatine
supplementation to a standard diet may eventually result in
a postexercise increment of muscle glycogen concentration
similar to that found after a classical carbohydrate-enriched
glycogen supercompensation dietary protocol (39). Interest-
ingly, after 5 weeks of creatine supplementation, the increase
of muscle glycogen content vanished, despite continued cre-
atine supplementation. In fact, during both immobilization and
rehabilitation, the pattern of muscle glycogen changes
closely mimicked the fluctuations of muscle total creatine con-
tent (Table 1) (Fig. 2). In this respect, Low et al. (40) have pro-
vided clear evidence that osmotic swelling of muscle cells is
a potent stimulus to muscle glycogen synthesis. The
30 mmol/kg DW increase of muscle total creatine, estab-
lished after 3 weeks of training in the creatine group, was
therefore probably sufficient to induce a degree of cell
swelling necessary to enhance insulin-stimulated glycogen
synthesis (40,36). If such an osmotic trigger mechanism
indeed regulates insulin action on glycogen synthesis during
creatine supplementation, then the decrease in muscle crea-
tine content beyond 3 weeks of training might also explain the
concurrent decrease in the muscle glycogen storage. The
mechanism behind the decrease in muscle creatine content
during the final stage of the study, despite continued creatine
ingestion at a rate presumed to be sufficient for maintaining
an elevated muscle creatine content (5 g/day), is unclear
(2,41). Studies in rats have demonstrated that long-term high-
dose creatine feeding induces a downregulation of muscle
total Na-creatine cotransporter protein content (42). In addi-
tion, the low creatine transporter content in failing human
myocardium has been found to be associated with a
decrease in intracellular creatine storage (43).
In conclusion, the current findings provide strong evidence
to suggest that 1) oral creatine supplementation can offset
the decline of muscle GLUT4 protein content in skeletal
musculature during disuse atrophy, and 2) oral creatine sup-
plementation increases GLUT4 content during subsequent
rehabilitation training. Based on the present findings, it is
warranted to evaluate the potential of long-term creatine
supplementation as a strategy to prevent or treat disease
conditions characterized by peripheral insulin resistance.
ACKNOWLEDGMENTS
This study was supported by grant G.0331.98 from the Fonds
voor Wetenschappelijk Onderzoek Vlaanderen, grant OT/94/31
from the Onderzoeksraad K.U.-Leuven, and grant 504-14 from
the Danish National Research Foundation.
The authors thank Betina Bolmgreen, Irene Beck Nielsen,
and Monique Ramaekers for providing skilled technical
assistance.
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