Regulation of Glycogen Synthase Kinase-3 in Human Skeletal Muscle
Effects of Food Intake and Bicycle Exercise
Jørgen F.P. Wojtaszewski,
Pernille Nielsen,
Bente Kiens, and
Erik A. Richter
From the Copenhagen Muscle Research Centre, Department of Human
Physiology, University of Copenhagen, Copenhagen, Denmark.
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ABSTRACT
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Studies of skeletal muscle from rodents performed both in vivo and in vitro
suggest a regulatory role of glycogen synthase kinase (GSK) 3 in glycogen
synthase (GS) activation in response to insulin. Recently, hyper-insulinemic
clamp studies in humans support such a role under nearly physiological
conditions. In addition, in rats the activation of GS in skeletal muscle
during treadmill running is time-related to the deactivation of GSK3. We
investigated whether GSK3 was deactivated in human muscle during low-
(
50% Vo2max for 1.5 h) and high-intensity (
75%
Vo2max for 1 h) bicycle exercise as well as food intake. We
observed a small but significant increase in GSK3
(10-20%) activity in
biopsies obtained from vastus lateralis after both low- and high-intensity
exercise, whereas GSK3ß activity was unaffected. Subsequent food intake
increased Aktphosphorylation (
2-fold) and deactivated GSK3
(
40%), whereas GSK3ß activity was unchanged. GS activity increased
in response to both exercise and food intake. We conclude that GSK3
but
not GSK3ß may have a role in the regulation of GS activity in response to
meal-associated hyperinsulinemia in humans. However, in contrast to findings
in muscle from rats, exercise does not deactivate GSK3 in humans, suggesting a
GSK3-independent mechanism in the regulation of GS activity in muscle during
physical activity.
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INTRODUCTION
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Glycogen synthase kinase (GSK) 3 is expressed in skeletal muscle and has
been identified in two isoforms,
and ß. Both isoforms contain
potential serine and tyrosine phosphorylation sites. Tyrosine phosphorylation
is important for full activation, but the regulatory role for these sites in
vivo is still unclear
(1,2,3).
Serine phosphorylation on sites 9 and 21 in GSK3ß and GSK3
,
respectively, is highly regulated in response to stimulation (e.g., by
insulin) and phosphorylation on these sites leads to deactivation of the
enzyme
(4,5,6).
In vitro GSk3 phosphorylates two of the four sites on glycogen synthase (GS),
which also become dephosphorylated in response to insulin
(7). This suggests that insulin
activates GS in part through a GSK3-dependent mechanism. Supporting this view
are our recent observations that the time course of serine phosphorylation and
deactivation of GSK3 and activation of GS is highly identical under both
supraphysiological insulin stimulation in the rat
(8) and during physiological
insulin clamp conditions in humans
(9). Interestingly, during
physiological conditions in humans only GSK3
is deactivated by insulin,
whereas supraphysiological insulin injection in the rat leads to deactivation
of both GSK3
and GSK3ß
(8,9,10).
In the period after exercise, glycogen resynthesis is a major metabolic
challenge for the skeletal muscle. Sustained activation of glucose uptake and
activation of GS together with enhanced insulin sensitivity for activation of
these processes eventually lead to normalization or even super-compensation of
the glycogen stores after exercise, as reviewed by Richter
(11). GS, the rate-limiting
enzyme for glycogen synthesis, displays enhanced activity ratio when isolated
from previously exercised muscle. This suggests that the enzyme becomes
dephosphorylated in response to exercise
(12), as reviewed by Ivy and
Kuo (13). Recently it was
reported that treadmill running leads to deactivation of GSK3
and
GSK3ß in rat skeletal muscle concurrent with GS activation
(8). This indicates that GSK3
may also regulate GS phosphorylation and activation in response to contractile
activity in rat skeletal muscle.
In the current investigation, we compared the effects of food intake and
high- as well as low-intensity exercise on the regulation of muscular GSK3 in
humans and compared this regulation with that of GS and the upstream kinase
Akt.
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RESEARCH DESIGN AND METHODS
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Subjects. Seven healthy men (25 ± 1 years) gave their
informed consent to participate in the study, which was approved by the
Copenhagen Ethics Committee. Body weight, height, and BMI were 74 ± 2
kg, 178 ± 2 cm, and 23 ± 1 kg/m2, respectively. The
subjects participated in regular physical activities twice a week on average
and were used to biking on a daily basis for local transportation. One to two
weeks before the experiments, maximal pulmonary oxygen consumption was
determined during an incremental bicycle ergometer test (55 ± 1 ml
· kg-1 · min-1).
Experimental protocol. The subjects randomly underwent two
experimental trials separated by 2-3 weeks. Subjects were instructed to eat a
controlled diet (carbohydrate
63%, fat
22%, and protein
15%,
average 13.5 MJ/day) for 3 days before each experiment and arrived at the
laboratory in the morning after an overnight fast. After 45 min of rest, a
needle biopsy from the vastus lateralis was obtained under local anesthesia. A
venous catheter was inserted in an antecubital vein for blood sampling. The
subjects then performed bicycle exercise for 90 min at 50% Vo2max
(low-intensity trial) or for 55 min at 75% Vo2max plus 5 min at 90%
Vo2max (high-intensity trial). The total energy expenditure was
aimed at being the same and did not differ in the two trials (average 3,800
kJ). Immediately after exercise, another biopsy was obtained from the vastus
lateralis. The subjects then rested in the supine or sitting position for 3 h,
having free access to water only. Then, a third biopsy was obtained, and the
subjects then ingested a carbohydrate-rich meal with an energy content similar
to the amount used during the exercise bout plus the expected energy
consumption in the 3-h postexercise period. Thus, this meal was identical in
the two trials, and consisted of
75% carbohydrate,
17% fat, and
8% protein, which amounted to 4,910 kJ in total. The meal was ingested
within 15 min. The subjects then rested in the supine or sitting position for
2 h before drinking a carbohydrate beverage, containing 500 kJ. Another hour
elapsed before a final biopsy was obtained from the vastus lateralis. The
biopsies before and after exercise were always taken in the same leg, and the
biopsies taken 3 and 6 h after exercise were always taken in the
contra-lateral leg. Incisions for biopsies were spaced 4-5 cm apart. Biopsies
were frozen in liquid nitrogen within 20 s. Before and during the last minute
of exercise as well as every following hour, blood samples were obtained for
measurements of plasma insulin concentrations.
Analytical procedures. Plasma insulin concentrations were measured
as previously described (14).
For determination of muscle glycogen content, muscle biopsies were
freeze-dried and dissected free of blood, fat, and connective tissue before
analysis. Glycogen content was determined as glycosyl units after acid
hydrolysis (15). Muscle GS
activity was measured in a homogenate by a modification of the method of
Thomas (16) as described by
Richter et al. (17). GS
activity was determined in the presence of 0.02, 0.17, and 8 mmol/l
glucose-6-phosphate (G6P) and given either as the percent of G6P-independent
GS activity (%I-form) (100 x activity in the presence of 0.02 mmol/l G6P
divided by the activity at 8 mmol/l G6P [saturated]) or as the percent of
fractional velocity (%FV) (100 x activity in the presence of 0.17 mmol/l
G6P divided by the activity at 8 mmol/l G6P).
As an indicator of Akt activation, Akt serine473 phosphorylation
was measured by Western blotting of muscle lysate using a phosphospecific
antibody (New England Biolabs, Beverly, MA) and fluorescent measurements of
alkaline phosphatase as a detection system (Promega U.K., Southampton, U.K.),
as previously described (14).
GSK3
and GSK3ß were immunoprecipitated from 100 µg of muscle
lysate using an anti-GSK3
(Upstate Biotechnology, Lake Placid, MA) or
anti-GSK3ß (Transduction Laboratories, San Diego, CA) antibody. A
p81-filter paper assay, with a phospho-GS2-peptide (Upstate Biotechnology) as
substrate, was used to measure GSK3 activity, as described previously
(8,9).
Calculations and statistics. Control samples were added to all
immunoblots and activity assays, and assay-to-assay variation was accounted
for by expressing the data relative to these samples. Data are expressed as
means ± SE. Statistical evaluation was done by one- or two-way analysis
of variance with repeated measures, as appropriate. When analysis of variance
revealed significant differences, a post hoc test was used to correct for
multiple comparisons (Student-Newman-Keuls test). Differences between groups
were considered statistically significant if P was <0.05.
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RESULTS
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Exercise and food intake effects on glycogen metabolism.
Figure 1 shows GS activity in
the low- and high-intensity exercise trials. Both %I-form and %FV of GS were
increased by exercise, and the increase tended to be higher in the
high-intensity trial. GS activity was not changed in the first 3 h after
exercise. Food intake raised GS activity further only in the high-intensity
trial, despite the fact that plasma insulin concentrations were elevated to a
similar extent in the two trials (Table
1). The rate of glycogen utilization was higher during high-
compared with low-intensity exercise (4.5 ± 0.7 vs. 1.6 ± 0.3
mmol/kg dry wt/min, P < 0.05). In the high-intensity trial,
glycogen content decreased further during the first 3 h after exercise, and
only in this trial was a significant (P < 0.05) glycogen
resynthesis (
25%) observed after food intake
(Fig. 2).

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FIG. 2. See Fig. 1 for details.
Glycogen content measured in freeze-dried muscle samples dissected free of
connective tissue, fat, and blood. *, Significant
differences (P < 0.05) from basal and 3 h, respectively. Data are
means ± SE, n = 6-7.
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Bicycle exercise in humans does not lead to deactivation of GSK3.
Glycogen-depleting exercise, performed at either a high or low intensity, did
not decrease the activity of GSK3
or GSK3ß
(Fig. 3). In fact, exercise
increased GSK3
activity in both trials in all subjects except one,
giving rise to a modest but significant (P < 0.05) increase in
both trials (
10-20%) (Fig.
3A). In accordance with this increase, Akt
serine473 phosphorylation decreased during exercise in the majority
of experiments (10 of 14) (Fig.
4), perhaps as a result of the significant (P < 0.05)
insulin-lowering effect of exercise (Table
1). However, overall the decrease in Akt phosphorylation did not
reach statistical significance.

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FIG. 4. See Fig. 1 for details.
Muscle Akt serine473 phosphorylation measured by phosphospecific
immunoblotting of muscle lysates. Significant differences (P
< 0.05) from all other measurements in the same trial. Data are means
± SE, n = 7.
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Increased plasma insulin levels after food intake lead to
phosphorylation of Akt and deactivation of GSK3
. Food intake was
associated with an increase in plasma insulin concentration, peaking 60 min
after food intake in both trials at an average of 85 µU/ml, followed by a
slight decrease 2 and 3 h after ingestion (
70 and
60 µU/ml,
respectively) (Table 1). Plasma
insulin concentrations were similar during the two trials at all time points.
Serine phosphorylation on site 473 of Akt increased by
2-fold in response
to food intake (Fig. 4). The
degree of phosphorylation was independent of the intensity of the prior
exercise bout. Likewise, GSK3
activity decreased significantly by
40% in response to food intake during both trials, whereas GSK3ß
activity was unchanged (Fig.
3).
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DISCUSSION
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The current results demonstrate that low- and high-intensity physical
exercise in humans is not associated with a deactivation of GSK3 in skeletal
muscle. In fact, the drop in plasma insulin concentration during exercise may
cause the minor, but significant, activation of GSK3
. Because GS is
highly activated during exercise, our data suggest that this activation in
response to exercise is not regulated by a GSK3-dependent mechanism in humans.
These findings are in line with our recent human data showing an elevated
muscle GS activity 4 h after exercise without any concurrent deactivation of
GSK3 (9). The lack of
deactivation of GSK3 with exercise in the present study contrasts with
observations in muscle from treadmill-exercised rats, in which an
exercise-induced decrease in both GSK3
activity and GSK3ß activity
was demonstrated (8).
For the first time, we show that a physiological stimulus such as intake of
food causes deactivation of muscle GSK3
in humans. Because deactivation
of GSK3
was recently demonstrated during hyperinsulinemic clamp
conditions
(9,10),
the GSK3
-deactivation observed in the present study is probably caused
by the meal-induced hyperinsulinemia, giving rise to increased Akt
phosphorylation (Fig. 4).
However, deactivation of GSK3ß is not observed
(Fig. 3B). During
euglycemic clamp conditions in healthy subjects and patients with type 2
diabetes, deactivation of GSK3ß was also not observed
([10] and unpublished
observation). In contrast, a marked deactivation of GSK3ß was observed in
rat skeletal muscle after injections of very large doses of insulin (10 U/kg
body wt) (8). The differences
between the findings in humans and in rats may relate to the species
differences or possibly to the level of hyperinsulinemia achieved, which was
clearly supraphysiological in the rats.
Another possibility is that insulin injections in rats result in
hypoglycemia giving rise to epinephrine secretion
(18). ß-adrenergic
stimulation of primary adipocytes decreased GSK3 activity immunoprecipitable
with an antibody recognizing both
and ß isoform of GSK3
(19). If ß-adrenergic
stimulation by epinephrine in response to hypoglycemia in the insulin-injected
rats also deactivates GSK3 in muscle, this could be the indirect mechanism by
which supraphysiological insulin concentrations cause deactivation of
GSK3ß. However, if so, we would expect similar effects of epinephrine
during exercise in humans because plasma epinephrine levels, especially during
the high-intensity exercise trial, are known to be elevated four- to sixfold
compared with rest (20). Thus,
more studies are needed to clarify the role of epinephrine in the regulation
of GSK3 activity in skeletal muscle.
Hyperinsulinemia as a result of food intake activates GS to a greater
extent after high-intensity exercise compared with low-intensity exercise, and
is accompanied by a significant glycogen resynthesis in the period after food
intake. Interestingly, this enhanced insulin action is not mediated through
changes in insulin signaling, as both Akt and GSK3 were affected similarly in
the two trials. Recently, we reported that enhanced insulin action in
exercised versus rested human skeletal muscle was also not a consequence of
enhanced intracellular insulin signaling
(9,14).
In fact, in those studies, a very strong correlation existed between the
degree of glycogen breakdown during the exercise bout and the subsequent rate
of muscle glucose uptake after insulin stimulation (r2 =
0.52, n = 14, P < 0.01 [unpublished observations]). In
the present study, a significant glycogen resynthesis occurred in response to
food intake in the high-intensity trial, in which a lower glycogen content was
also evident after exercise. In fact, good negative correlations exist between
the glycogen level before food intake and the following: 1) the
increase in glycogen content after food intake (r2 = 0.43,
n = 13, P < 0.01) and 2) the increase in GS
activity after food intake (e.g., for %I-form r2 = 0.31,
n = 13, P < 0.05)
(Fig. 5). These observations
are in line with other recent observations indicating that muscle glycogen
content is important for the ability of both insulin and contractions to
stimulate muscle glucose uptake in rats
(21,22,23).
In contrast to the present study in humans, exercise in rats leads to
deactivation of GSK3
and GSK3ß in muscle
(8). The explanation of this
difference is not easily found, but it cannot be excluded that even more
severe exercise than in the present study, leading to more severe glycogen
depletion, could cause deactivation of GSK3 also in human muscle.
Nevertheless, our findings that two rather strenuous exercise bouts consuming
close to 4 MJ, leading to marked glycogen depletion and GS activation, do not
lead to GSK3 deactivation suggests that GSK3 deactivation during exercise is
not physiologically important for activation of GS in human skeletal muscle. A
putative alternative mechanism for dephosphorylation of GS in response to
exercise could be an activation of protein phosphatases (PPs), especially PP1
(24). PP1 is targeted to
glycogen through binding to regulatory binding proteins. For example, protein
targeting to glycogen (PTG) has been shown to form complexes to the catalytic
subunit of PP1 and to glycogen. In addition, there may be an association with
other enzymes involved in glycogen metabolism
(25). Thus, regulation of the
binding properties of these binding proteins may be important for the
regulation of GS activity in response to exercise. In fact, a very recent but
preliminary report indicates that in the absence of the regulatory glycogen
binding subunit GM (or RGL) of PP1, a marked reduction
in GS activation in response to contractile activity is observed in mouse
skeletal muscle (26). To our
knowledge, only one study has measured muscular PP1 activity in response to
exercise. Thus, in humans PP1 activity actually decreases during maximal
isometric muscle contractions and returns to pre-exercise levels early in the
recovery period (27). Whereas
these findings do not offer any explanation for the postexercise-increased GS
activity after isometric contractions, they do not exclude a regulatory role
of PP1 during more dynamic glycogen-depleting exercise as in the present
study.
In conclusion, food intake is associated with muscular deactivation of
GSK3
, supporting a physiological role for GSK3
in the regulation
of GS activity in human skeletal muscle. However, the regulation of GS in
response to food intake (elevated plasma insulin) is not solely dependent on
GSK3
deactivation because muscle glycogen content clearly has a
signaling-independent influence on the effects of food intake on insulin
action. Finally, in contrast to rodents, moderate and intense exercise in
humans does not lead to GSK3 deactivation in skeletal muscle, excluding the
kinase as an important regulator of GS activity during physical activity in
humans.
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ACKNOWLEDGMENTS
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This study was supported by grant no. 504-14 from the Danish National
Research Foundation. J.F.P.W. was supported by a postdoctoral fellowship from
the Danish Medical Research Council.
We are grateful to Betina Bolmgren, Winnie Taagerup, and Irene B. Nielsen
for superior technical assistance.
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FOOTNOTES
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%FV, percent of fractional velocity; %I-form, percent of G6P-independent GS
activity; G6P, glucose-6-phosphate; GS, glycogen synthase; GSK, GS kinase; PP,
protein phosphatase.
Address correspondence and reprint requests to Jørgen F.P.
Wojtaszewski, Copenhagen Muscle Research Centre, August Krogh Institute,
University of Copenhagen, 13, Universitetsparken, DK-2100 Copenhagen, Denmark.
Email:
jwojtaszewski{at}aki.ku.dk
.
Received for publication June 28, 2000
and accepted in revised form October 24, 2000
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