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
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{at}flok.kuleuven.ac.be
.
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ABSTRACT
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The purpose of this study was to investigate the effect of oral creatine
supplementation on muscle GLUT4 protein content and total creatine and
glycogen content 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-extensor 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
offsets 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.
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INTRODUCTION
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It 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
output 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 supplementation to treat muscle atrophy and
neuromuscular diseases. Thus, the recovery of muscle disuse atrophy due to
immobilization was significantly enhanced by creatine supplementation (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). 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
(5,6,7).
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 transport 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 concentrations obtained by oral creatine
intake in humans do not affect insulin secretion
(14,15).
Perhaps the most striking evidence 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 hyperglycemia 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 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 inactivity and training are effective stimuli to down-
and upregulate muscle GLUT4 content and peripheral insulin sensitivity,
respectively (17). Therefore,
we investigated the effect of creatine 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 investigated the effects of creatine supplementation on muscle
functional capacity during disuse atrophy in healthy subjects.
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RESEARCH DESIGN AND METHODS
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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 physical activity level and other living habits during
the period of the study. However, 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 creatine or a placebo group based on quadriceps muscle
cross-sectional area and maximal isometric knee-extension torque to obtain two
groups of similar distribution (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 rehabilitation program. Each training session
consisted of four series of 12 unilateral 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 investigators. During
immobilization, the creatine group received 5 g creatine monohydrate four
times per day, whereas the placebo group received placebo supplements (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 creatine) 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% carbohydrate, 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% lidocaine). 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 connective tissue, rapidly frozen
in liquid nitrogen, and stored at -80°C for subsequent 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 (Polytron) for 30 s on ice in a buffer with the following
composition: 150 mmol/l NaCl, 1% NP4O, 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 Trisbuffered 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.
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RESULTS
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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 rehabilitation 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 concentration 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 phosphocreatine 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
concentration was not significantly changed compared with the baseline value
during either immobilization or rehabilitation. Yet 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
rehabilitation (P < 0.05). However, along with the declining
muscle phosphocreatine levels, muscle total creatine returned to baseline by
the end of the study.
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DISCUSSION
|
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Our study investigated the impact of creatine supplementation on muscle
GLUT4 content and glycogen and total creatine concentrations in healthy
subjects during 2 weeks of voluntary leg immobilization followed by 10 weeks
of rehabilitation 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 initial 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 glucose uptake.
Conversely, reducing the content of GLUT4 by GLUT4 knockout, denervation, or
aging impairs insulin-mediated muscle glucose uptake
(19). Our data, therefore,
suggest that creatine supplementation in humans may increase insulin
sensitivity by increasing muscle GLUT4 content.
Over the last decade, substantial evidence has accumulated to show that
endurance exercise training elevates muscle GLUT4 content and
insulin-stimulated glucose uptake in both healthy
(17,20,21,22,23,24,25,26,27,28)
and insulin-resistant muscles
(29,30).
In this respect, the current study shows that in healthy individuals, a low
volume (3 weekly sessions) of moderate resistance training (60% of 1
repetition maximum [RM]), in contrast with endurance training
(23,24,25,26,
28) or daily maximal
resistance 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 supplementation can
probably increase GLUT4 protein content in skeletal musculature independent of
exercise training. In keeping with earlier observations
(17,20,21,22,31,32),
muscle deconditioning 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 supplementation in the absence of a training stsore, it is reasonable
to conclude that creatine supplementation can increase GLUT4 protein 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 immobilization 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 conditions 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 creatine-induced increase in AMPK activity has not been found.
Alternatively, there is substantial evidence to suggest that cellular
hydration status is an important factor controlling cellular protein turnover
(36), which in muscle cells,
excluding the contractile 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 synthesis and degradation in muscle cells.
The bulk of glucose in the human body is stored as muscle glycogen. The
presence of a high muscle glycogen concentration, in general, indicates
adequate insulin stimulation of muscle glucose uptake and glycogen synthesis.
Furthermore, 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, 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).
Interestingly, after 5 weeks of creatine supplementation, the increase of
muscle glycogen content vanished, despite continued creatine supplementation.
In fact, during both immobilization and rehabilitation, the pattern of muscle
glycogen changes closely mimicked the fluctuations of muscle total creatine
content (Table 1) (Fig. 2). In this respect, Low
et al. (40) have provided
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, established 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 creatine 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 addition, 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 supplementation 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.
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ACKNOWLEDGMENTS
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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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FOOTNOTES
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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.
Received for publication September 13, 2000
and accepted in revised form October 24, 2000
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