From the School of Biochemistry and Genetics, The
Medical School, University of Newcastle upon Tyne, NE2 4HH, United
Kingdom, the ** Adfeling Biochemie, Faculteit Geneeskunde, Katholieke
Universiteit Leuven, B-3000 Leuven, Belgium,
Novo Nordisk,
DK-2880, Bagsvaerd, Denmark, and the
§§ Friedrich Miescher-Institut, Maulbeerstrasse
66, CH-4056 Basel, Switzerland
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ABSTRACT |
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The regulation of glycogen synthesis and
associated enzymes was studied in human myoblasts and myotubes
maintained in culture. Both epidermal growth factor (EGF) and insulin
stimulated glycogen synthesis approximately 2-fold, this stimulation
being accompanied by a rapid and stable activation of the controlling
enzyme glycogen synthase (GS). EGF also caused inhibition of glycogen
synthase kinase 3 (GSK-3) and activation of the A key step in the lowering of blood glucose levels by insulin is
the promotion of uptake of glucose into muscle and its subsequent storage as glycogen. This involves recruitment of additional glucose transporters to the plasma membrane and stimulation of the enzyme glycogen synthase (GS)1
(reviewed in Ref. 1). The activity of GS is regulated by reversible phosphorylation at a number of sites. The key sites involved in the
regulation of GS are collectively referred to as sites 3, phosphorylation of which by glycogen synthase kinase 3 (GSK-3) leads to
inactivation of GS (2). The dephosphorylation and re-activation of GS
is catalyzed by a glycogen-bound form of protein phosphatase 1 (PP1G) (3).
Insulin activates GS via dephosphorylation of the protein. There is
evidence for insulin exerting this stimulatory effect on GS by both
activating PP1G and by inhibiting GSK-3. Recently, attention has focused on the mechanism by which insulin inhibits GSK-3,
with a plausible scheme emerging to link events at the plasma membrane
with regulation of GSK-3. GSK-3 is inactivated by phosphorylation at a
single serine residue close to its amino terminus (4). At least three
insulin-stimulated protein kinases, namely
p70S6k, p90S6k,
and protein kinase B (PKB) are capable of catalyzing this
phosphorylation in vitro. Each of these kinases lies
downstream of an insulin-stimulated cascade (5). However, current
evidence indicates that PKB is responsible for this insulin-stimulated
phosphorylation of GSK-3 in a variety of cell types (5-7). PKB is
itself phosphorylated and activated in response to insulin, this being
mediated, at least in part, by 3-phosphoinositide-dependent
protein kinase 1 (PDK1) (8-9). The phosphorylation of PKB by PDK1 is
dependent on the presence of phosphatidylinositol (3-5) triphosphate
(10), the major lipid product of PI-3 kinase, an enzyme activated by insulin via binding to insulin receptor substrate 1 at the plasma membrane (reviewed in Ref. 11).
In addition to insulin, several other growth factors and hormones
influence glycogen synthesis and the signaling pathways involved. Of
particular interest is the action of epidermal growth factor (EGF)
which apparently has different effects on glycogen metabolism in
different cell types. In isolated rat adipocytes, EGF stimulates
neither glycogen synthesis nor GS activity, despite stimulating
p70S6k and
p90S6k (12). Consistent with the failure to
activate GS is the observation that, unlike insulin, EGF has only a
small and transient effect on the activities of PKB and GSK-3 (13). In
contrast, EGF inhibits GSK-3 in hepatocytes to the same extent as
insulin (14) and yet it does not stimulate glycogen synthesis, indeed
it antagonizes the stimulatory effect of insulin on this metabolic
effect (15). This antagonistic effect is not however observed in
adipocytes (12).
It is well established that insulin inactivates GSK-3 and activates GS
in a variety of muscle systems (5, 6, 13, 16, 17) but little work has
been carried out using EGF, with the exception of one study in rat
diaphragm which reported that EGF does not stimulate GS (18). This
laboratory has utilized cultured human muscle cells to study the
control of glycogen synthesis by insulin (6, 17). Here we report that
EGF stimulates GS and glycogen synthesis in both human myoblasts and
myotubes and that this is apparently mediated via the PKB/GSK-3 pathway.
Materials--
All tissue culture trays were from Costar
(Cambridge, MA). Culture media, penicillin/streptomycin, and
trypsin-EDTA were from Life Technologies, Inc. (Paisley, UK). Chick
embryo extract was obtained from ICN (Costa Mesa, California).
Anti-GSK-3 Cell Culture--
Human myoblasts were grown from needle biopsy
samples taken from the gastrocnemius muscle of healthy subjects with no
family history of Type 2 diabetes and with normal glucose tolerance and normal insulin sensitivity as assessed using the short insulin tolerance test.
Myoblasts were maintained in growth medium consisting of Ham's F-10
nutrient mixture containing 20% fetal calf serum, 1% chick embryo
extract, 10,000 units/ml penicillin, and 2 mg/ml streptomycin. Myoblasts were fused to form myotubes by incubation for up to 21 days
in Estimation of Glycogen Synthesis--
Glycogen synthesis was
determined as 14C-glucose incorporation into glycogen.
Cells were incubated for 1 h in culture medium containing
[U-14C]glucose (5.5 mM glucose; 1.25 µCi/ml) and either insulin, EGF, or both. Medium was then removed,
and the cells were rapidly washed five times in ice-cold
phosphate-buffered saline. Cells were lysed by the addition of 20% w/v
KOH that was neutralized after 1 h by the addition of 1 M HCl. The wells were aspirated and washed with 400 µl of
distilled water. The aspirates were boiled for 5 min, and glycogen was
added to give a final concentration of 12 mg/ml. Glycogen was
precipitated with ethanol at 0 °C for 1 h. The samples were
centrifuged at 1700 × g for 10 min, pellets were
redissolved in formic acid, and radioactivity was determined by
scintillation counting. Results were expressed as pmol of glucose incorporated into glycogen per minute, per milligram of cell protein.
Preparation of Cell Extracts for Assay of Glycogen
Synthase--
Following the indicated treatments, cells were rapidly
washed three times with ice-cold PBS and collected, by scraping, into GS extraction buffer (10 mM Tris-HCl, pH 7.8, 150 mM KF, 15 mM EDTA, 60 mM sucrose, 1 mM 2-mercaptoethanol, 10 µg/ml leupeptin, 1 mM benzamidine, 1 mM phenylmethylsulfonyl
fluoride). Cells were then disrupted by sonicating for 8 s, using
a Soniprep 150. Homogenates were centrifuged at 13,000 × g for 10 min and the pellets discarded. Glycogen synthase
activity was assayed as incorporation of 3H-glucose from
uridine-5'-diphosphate [U-3H]glucose into glycogen, as
described in (19). Samples were incubated with reaction mixture (50 mM Tris-HCl, pH 7.8, 20 mM EDTA, 25 mM KF, 1% glycogen, 0.4 mM
UDP-[3H]glucose (specific activity 3000 dpm/nmol)),
containing either 0.1 mM (active), or 10 mM
(total) glucose-6-phosphate, for 30 min at 30 °C (19). Results were
expressed as fractional activities (active/total).
Preparation of Cell Extracts for Protein Kinase
Assays--
Following incubation with appropriate hormones, growth
factors, or other agents, cells were washed three times with ice-cold phosphate-buffered saline, and excess liquid was removed. Cells were
then scraped into kinase extraction buffer (100 mM
Tris-HCl, pH 7.4, containing 100 mM KCl, 2 mM
EDTA, 25 mM KF, 0.1% (v/v) Triton X-100, 1 mM
benzamidine, 0.1 mM Na3VO4, 1 µg/ml pepstatin, 1 µg/ml antipain, and 1 µg/ml leupeptin),
transferred to 1.5-ml Eppendorf tubes, and immediately frozen in liquid
nitrogen. Samples were subsequently thawed, sonicated for 1 min
(Sonibath; Dawe Ultrasonic Ltd., London, UK), and centrifuged at
13,000 × g for 5 min at 4 °C. GSK-3 or PKB were
immunoprecipitated from aliquots of the supernatant containing
approximately 10 µg of protein. In each case, immunoprecipitations
was carried out using appropriate antibodies that had been pre-adsorbed
to Pansorbin for 2 h at 4 °C. Following incubation of extracts
with the antibody/Pansorbin complex for 1.5 h at 4 °C, the
immune complex was recovered by centrifugation for 3 min at 13,000 × g. The supernatant was removed and the complex washed
once with kinase extraction buffer and twice with buffer A (50 mM sodium glycerophosphate, pH 7.4, containing 1 mM EGTA, 1 mM benzamidine, 1 mM
dithiothreitol, 0.1 mM Na3VO4, and
1 mg/ml each of pepstatin, antipain, and leupeptin).
GSK-3 activity in immunoprecipitates was assayed in a final volume of
20 µl containing 25 mM sodium glycerophosphate, pH 7.4, 100 mM NaCl, 50 µM phospho-GS peptide
substrate (termed 2B-(SP)) (20), 50 µM
[ In preliminary experiments, the effect of varying concentrations
of EGF on glycogen synthesis in myoblasts was investigated. Incubation
of cultured myoblasts with EGF (100 nM) caused a maximal increase in the incorporation of glucose into glycogen from 193 ± 18 to 360 ± 49 pmol/min/mg (n = 7, p < 0.05, compared with control). This increase was
similar to that evoked by insulin which stimulated the rate of glycogen
synthesis to 462 ± 55 pmol/min/mg (n = 7 independent experiments in cells from three subjects, p < 0.05, compared with control).
Incubation with EGF also caused a time-dependent increase
in the activity ratio of GS, with stimulation being observed within 5 min and reaching a maximum of approximately 2-fold after 10-15 min
(Fig. 1a). The magnitude and
time courses are similar to those observed previously in response to
insulin (6) although the basal values in the present work are
significantly lower than reported previously. No synergistic effect was
observed when the two agonists were added simultaneously (Fig.
1b). Similar effects were observed in myotubes, where EGF
(100 nM) caused a significant increase in the activity
ratio of GS from 0.014 ± 0.0012 to 0.030 ± 0.0024 (n = 6 independent experiments in cells from three
subjects, p < 0.05, compared with control).
isoform of protein
kinase B (PKB) with the time-course and magnitude of its effects being similar to those induced by insulin. An inhibitor of the
mitogen-activated protein (MAP) kinase pathway did not prevent
stimulation of GS by EGF, suggesting that this pathway is not essential
for the effect. A partial decrease in the fold activation of GS was,
however, observed when p70S6k activation
was blocked with rapamycin, suggesting a contribution of this pathway
to the control of GS by either hormone. Wortmannin, a selective
inhibitor of phosphatidylinositol 3'-kinase (PI-3 kinase) completely
blocked the effects of both EGF and insulin in these cells. These
results demonstrate that EGF, like insulin, activates glycogen
synthesis in muscle, acting principally via the PKB/GSK-3 pathway but
with a contribution from a rapamycin-sensitive component that lies
downstream of PI-3 kinase.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
and anti-GSK-3
antibodies and antibodies to the
PH-domain of PKB
were as described previously (16, 5). [
-32P]ATP (148 TBq/mmol) was obtained from ICN.
D-[U-14C]glucose (10 GBq/mmol) and
UDP-[6-3H]glucose (252 GBq/mmol) were from Amersham
Pharmacia Biotech (Buckinghamshire, UK). Wortmannin and rapamycin were
from Sigma (Poole, UK), and PD98059 was from New England Biolabs
(Massachusetts). Actrapid insulin was from Novo Nordisk (Copenhagen,
Denmark). Mouse EGF was from Sigma.
-minimal essential medium containing 2% fetal calf serum, 2 mM L-glutamine, 10,000 units/ml penicillin, and
2 mg/ml streptomycin. All experiments were performed using cells
between the fifth and fifteenth passage at greater than 90%
confluence. Prior to hormone treatment, cells were incubated for at
least 2 h in serum-free medium.
-32P]ATP (approximately 4000 cpm/pmol), 10 mM MgCl2, 0.5 mM benzamidine, 0.5 mM dithiothreitol, 0.05 mM
Na3VO4, and 2.5 mM inhibitory
peptide of cyclic AMP-dependent protein kinase (PKI) (6).
After incubation for 30 min at 30 °C, samples were centrifuged at
13,000 × g for 3 min, and 15 µl of the supernatant
containing the radiolabeled peptide product was spotted onto
1-cm2 Whatman P81 phosphocellulose paper squares. After
washing in 175 mM phosphoric acid with four changes, the
papers were dried and phosphate incorporation was determined by liquid
scintillation counting. PKB was assayed in an identical manner except
that activity was measured against the PKB substrate Crosstide (100 µM) (5). One milliunit enzyme of activity was defined as
that which catalyzes the incorporation of 1 nmol of phosphate into
peptide substrate in 1 min.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Activation by EGF and insulin of glycogen
synthase in human myoblasts. Glycogen synthase activity was
measured in the presence of low and high concentrations of
glucose-6-phosphate (G6P), as described under " Experimental Procedures." In panel a, cells were treated
with 100 nM EGF for the times shown prior to extraction.
The resulting activity ratios represent the mean ± S.E. of
n = 4, in two different subjects. Statistical
significance (p < 0.05) compared with basal values is
indicated by *. In panel b, cells were incubated
with or without 100 nM insulin,100 nM EGF, or
both for 10 min. The resulting activity ratios represent the mean ± S.E. of n = 11, in five different subjects.
Statistical significance (p < 0.05) compared with
basal values is indicated by *.
Previous work from several laboratories has indicated that the activation of GS by insulin is mediated via inactivation by GSK-3. The effect of EGF and insulin on the activity of GSK-3 is shown in Fig. 2. EGF causes a rapid decrease in the activity of GSK-3 (Fig. 2a) and both the magnitude (Fig. 2b) and time course (6) of this effect are similar to those observed with insulin.
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To probe the events upstream of GSK-3, selective inhibitors of
p70S6k activation (rapamycin),
p90S6k activation (PD98059), and PI-3
kinase (wortmannin) were utilized. Fig. 3
demonstrates that inactivation of GSK-3 by EGF is not significantly affected by rapamycin or PD98059 but is blocked completely by wortmannin. These observations are consistent with those for insulin (5, 6), implicating PKB as the kinase responsible for phosphorylation and inactivation of GSK-3 in response to EGF. This is supported by the
data in Fig. 4, which shows that PKB
is rapidly activated by EGF, the activation being sufficiently rapid to
account for the time course of GSK-3 inactivation (Fig. 4a).
Once again the magnitude of activation in response to EGF was similar
to that evoked by insulin (Fig. 4b).
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The effect of these inhibitors on the activity of GS was also examined (Table I). Whereas rapamycin had little effect on the basal activity of GS, both PD98059 and wortmannin caused a decrease in the fractional activity in the absence of agonists. Upon addition of EGF, full-fold activation was observed in the presence of PD98059, implying that the MAP kinase p90S6k pathway is not essential for the activation of GS by EGF, despite EGF being a potent stimulator of that pathway (12). Wortmannin completely blocked activation of GS by EGF. The EGF-induced stimulation of GS activity was, however, reduced in the presence of rapamycin (EGF alone at 1.8-fold versus EGF/rapamycin at 1.3-fold). We have previously reported a similar effect of rapamycin on the activation of GS following insulin treatment of these cells (6).
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DISCUSSION |
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Human muscle cells in culture represent an appropriate model system for the study of cell signaling. Myoblasts and myotubes in culture show many properties of mature muscle in terms of their responsiveness to insulin and other agonists, but they are immature cells and show several important differences from mature muscle (6, 17). One prime example is that myoblasts do not express significant levels of the insulin-responsive glucose transporter Glut-4 and show little increase in glucose uptake in response to insulin (21). Although this is a limitation of the cells, it does have the advantage that observed effects on glycogen synthesis are effectively independent of effects on glucose uptake and therefore presumably because of changes in the activity ratio of GS. Hence, although the cells represent a good model for study of events in human muscle in vivo, some of the data must be interpreted with caution when extrapolating to the situation in vivo, although these concerns apply even more when using immortalized cell lines taken from a variety of animal species.
The data reported here indicate that, in myoblasts obtained from healthy human volunteers, EGF causes an approximate 2-fold stimulation of the rate of incorporation of extracellular glucose into intracellular glycogen. This is associated with a relatively rapid and sustained activation of glycogen synthase. In both cases, the extent of stimulation and the time course of the effects were similar to these observed in response to insulin (6).
Several observations suggest that common pathways are involved in the stimulation of GS by both EGF and insulin. First, both the time course and extent of the effects of the two agonists were remarkably similar for all the parameters examined. Second, the stimulation of GS activity by EGF and insulin was not additive if both agonists were present simultaneously. Finally, rapamycin was able to partially prevent the stimulation of GS by both EGF and insulin. Overall, the data suggest that the stimulation of glycogen synthesis in response to both EGF and insulin is mediated principally via the PKB/GSK-3 pathway, leading to the dephosphorylation of GS and stimulation of its activity, but with an additional contribution via a rapamycin-sensitive pathway that does not involve inhibition of GSK-3. Previous work on 3T3-L1 adipocytes has demonstrated involvement of a rapamycin-sensitive component in the stimulation of glycogen synthesis and GS by insulin (22). It has been reported previously, using A431 cells, that the phosphorylation and inactivation of GSK-3 in response to EGF is not blocked by rapamycin, effectively ruling out a role for p70S6k in that event (23). This is supported by the present work (Fig. 3) which demonstrates that rapamycin does not block the EGF-induced inhibition of GSK-3. Similarly PD98059 is without significant effect on this inhibition (Fig. 3), implying that the MAP kinase signaling pathway is not directly involved. We have shown previously that rapamycin and PD98059 had no effect on the inhibition of GSK-3 in response to insulin (6).
The finding of different effects of EGF on glycogen synthesis in
different cell systems remains enigmatic (12-15). One possible explanation is that some cells may express a counter-regulatory pathway, triggered by EGF which acts to antagonize the stimulatory effects on GS and glycogen synthesis. Of particular interest is the
comparison of the present data in muscle with observations with
isolated rat hepatocytes where EGF inactivates GSK-3 to the same extent
as insulin and also causes a significant transient activation of PKB
(14) and yet does not stimulate glycogen synthesis and indeed
antagonizes the stimulatory effect of insulin on that metabolic
parameter (15). It is clear, however, that EGF has a range of effects
in hepatocytes, which possess both high and low affinity forms of the
EGF receptor (24). The effects of EGF on glycogen synthesis in
hepatocytes are complex in that they are dependent on conditions of
cell culture (e.g. cell density) and the morphology of the
cells. Furthermore, there appears to be a pertussis toxin-sensitive
component to the action of EGF (15). The ability of EGF to activate
phospholipase C in hepatocytes (25) leading to rises in the cytosolic
levels of Ca2+ may also trigger a counter-regulatory
response, which overrides the stimulatory effects of EGF (and insulin)
on glycogen synthesis. In contrast, the major if not sole effect of EGF
in muscle is to stimulate glycogen synthesis, via activation of GS. The
stimulatory effects of EGF are observed in the absence of insulin and
are not augmented by insulin, implying a common pathway and providing no evidence for an additional EGF-specific pathway.
It has been previously reported that EGF fails to stimulate the activity of GS in isolated rat diaphragm (18). The finding that EGF also fails to stimulate p70S6k in that system is consistent with the possibility that a rapamycin-sensitive pathway is the major pathway controlling GS by EGF, under the conditions studied in that system. The relative importance of the respective signaling pathways in human muscle in vivo remains to be determined.
The observation (Table I) that the MAP kinase/ERK kinase inhibitor PD98059 causes a decrease in the activity state of GS under basal conditions implies that the MAP kinase pathway is involved in the maintenance of GS activity under basal conditions in these cells. This is consistent with our previous observation that considerable p90S6k activity is present in human myoblasts in the absence of any known agonist (6). However, upon addition of EGF, the activity of GS rises significantly, and indeed, the increase in the presence of PD98059 is equivalent (in fold terms) to the increase induced by EGF in the absence of the inhibitor. It is possible that the effect of PD98059 on the basal values of GS is because of the involvement of the MAP kinase pathway in the regulation of a GS phosphatase, possibly PP1G (3). Thus, appreciable levels of MAP kinase activity in the basal state may partially activate the GS phosphatase, whereas inhibition of MAP kinase activity with PD98059 would inactivate the phosphatase, suppressing GS activity. This scenario would allow for subsequent stimulation of GS via inhibition of GSK-3 alone, a process which is unaffected by PD98059. Similarly, lowering of the basal activity ratio of GS by wortmannin implies a role for PI-3 kinase and the MAP kinase pathway (16) in maintaining the basal state of activity.
In summary, the data reported here demonstrate that EGF stimulates both
glycogen synthesis and GS in human muscle cells in culture. This is
mediated primarily via the GSK-3/PKB pathway, but there is also a
contribution to the activation of GS from a component which is
rapamycin-sensitive and lies downstream of PI-3 kinase.
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ACKNOWLEDGEMENTS |
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We thank Dorothy Fittes for major practical contribution to the tissue culture work and Prof. D. M. Turnbull for invaluable advice.
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FOOTNOTES |
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* This work was supported in part by a grant from the Medical Research Council, UK.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a CASE studentship from the Biotechnology and Biological Sciences Research Council, UK, partly funded by Novo Nordisk.
¶ Recipient of a postgraduate studentship from the British Diabetic Association.
Is a Research Director of the Fonds voor Wetenschappel
k
Onderzoek-Vlaanderen.
¶¶ To whom correspondence should be addressed. Tel.: 44-191-222-7433; Fax: 44-191-222-7424; E-mail: S.J.Yeaman{at}ncl.ac.uk.
The abbreviations used are: GS, glycogen synthase; GSK-3, glycogen synthase kinase 3; PP1G, glycogen-bound form of protein phosphatase 1; PKB, protein kinase B; PDK1, 3-phosphoinositide-dependent protein kinase 1; EGF, epidermal growth factor; MAP, mitogen-activated protein.
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REFERENCES |
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