(Received for publication, October 12, 1995; and in revised form, November 27, 1995)
From the
Incubating rat diaphragm muscles with insulin increased the
glycogen synthase activity ratio (minus glucose 6-phosphate/plus
glucose 6-phosphate) by approximately 2-fold. Insulin increased the
activities of mitogen-activated protein (MAP) kinase and the M = 90,000 isoform of ribosomal protein S6
kinase (Rsk) by approximately 1.5-2.0-fold. Epidermal growth
factor (EGF) was more effective than insulin in increasing MAP kinase
and Rsk activity, but in contrast to insulin, EGF did not affect
glycogen synthase activity. The activation of both MAP kinase and Rsk
by insulin was abolished by incubating muscles with the MAP kinase
kinase (MEK) inhibitor, PD 098059; however, the MEK inhibitor did not
significantly reduce the effect of insulin on activating glycogen
synthase. Incubating muscles with concentrations of rapamycin that
inhibited activation of p70
abolished the activation of
glycogen synthase. Insulin also increased the phosphorylation of PHAS-I
(phosphorylated heat- and acid-stable protein) and promoted the
dissociation of the PHAS-I
eIF-4E complex. Increasing MAP kinase
activity with EGF did not mimic the effect of insulin on PHAS-I
phosphorylation, and the effect of insulin on increasing MAP kinase
could be abolished with the MEK inhibitor without decreasing the effect
of insulin on PHAS-I. The effects of insulin on PHAS-I were attenuated
by rapamycin. Thus, activation of the MAP kinase/Rsk signaling pathway
appears to be neither necessary nor sufficient for insulin action on
glycogen synthase and PHAS-I in rat skeletal muscle. The results
indicate that the effects of insulin on increasing the synthesis of
glycogen and protein in skeletal muscle, two of the most important
actions of the hormone, involve a rapamycin-sensitive mechanism that
may include elements of the p70
signaling pathway.
Glycogen synthesis in skeletal muscle has a key role in the
control of blood glucose levels by insulin. The large majority of
postprandial glucose uptake occurs in skeletal
muscle(1, 2) , and most of the glucose that enters
muscle fibers in response to insulin is converted to
glycogen(3) . This hormonal effect involves activation of
glycogen synthase, the enzyme that catalyzes the rate-limiting step in
the conversion of intracellular glucose to
glycogen(4, 5) . Insulin activates synthase by
promoting dephosphorylation of sites in the COOH- and
NH-terminal regions of the enzyme(4, 5) .
The pattern of dephosphorylation is consistent with the hypothesis that
insulin activates PP1
, the glycogen-bound form of type I
protein phosphatase, as this phosphatase is able to dephosphorylate
multiple sites in the synthase subunit(6) . PP1
is
controlled by phosphorylation of sites in its regulatory
subunit(6, 7) , designated R
(8) .
Phosphorylation of site 1 increases phosphatase activity toward
glycogen synthase. This site is readily phosphorylated in vitro by Rsk-2(9, 10, 11) , a kinase that is
phosphorylated and activated by MAP (
)kinase when cells or
tissues are incubated with insulin(12, 13) . Injecting
insulin into rabbits has been reported to increase phosphorylation of
site 1 in R
(9) , and it is widely believed that
the activation of glycogen synthase by insulin in skeletal muscle
involves the sequential activation of MAP kinase, Rsk-2, and
PP1
.
Insulin also stimulates protein synthesis in many cells, but again skeletal muscle is of particular importance as this tissue is the largest reservoir of body protein(14) . Muscle wasting is a hallmark of untreated diabetes mellitus in humans, and inducing diabetes in rats decreases by half the rate of protein synthesis in skeletal muscle (15) . Experimental diabetes also causes dispersion of polysomes and accumulation of free ribosomal subunits(16, 17) . Within 2 h of treating diabetic rats with insulin, the polysome profile returns to the prediabetic state. These effects of insulin and diabetes are indicative of regulation of translation initiation, which is generally the rate-limiting phase of protein synthesis(18, 19, 20) . Initiation involves recognition of capped mRNA, melting of secondary structure in the 5`-nontranslated region of the mRNA, and binding of the 40 S ribosomal subunit. Initiation is mediated by several factors, the least abundant of which is the mRNA cap-binding protein, eIF-4E. Several lines of evidence indicate that eIF-4E activity is limiting for initiation(18, 19, 20) .
Insulin increases
eIF-4E activity in adipocytes by stimulating the phosphorylation of the
translational regulator,
PHAS-I(21, 22, 23, 24) . PHAS-I cDNA
was originally cloned from a rat skeletal muscle library, and PHAS-I
mRNA was found in highest levels in skeletal muscle and fat (25) . Nonphosphorylated PHAS-I binds tightly to eIF-4E and
inhibits translation(24) , probably by preventing the
association of eIF-4E with eIF-4(26) . When PHAS-I is
phosphorylated in response to insulin, the PHAS-I
eIF-4E complex
dissociates(21, 22, 23, 24) ,
allowing eIF-4E to participate in translation initiation. PHAS-I is an
excellent substrate for MAP kinase in vitro, and the major
site (Ser
) phosphorylated by MAP kinase in vitro is phosphorylated in response to insulin in
adipocytes(27) . Moreover, essentially all of the
insulin-stimulated PHAS-I kinase activity in adipocyte extracts is
accounted for by the ERK-1 and ERK-2 isoforms of MAP
kinase(23) . Based on these results, MAP kinase was proposed to
mediate the phosphorylation of PHAS-I by insulin in adipocytes. Thus
far, all studies of the regulation of PHAS-I by insulin have been
confined to adipocytes.
Recent findings indicate that activation of
MAP kinase is neither necessary nor sufficient for the effects of
insulin on glycogen synthase in fat
cells(28, 29, 30, 31, 32) .
EGF and other agents that are as effective as insulin in activating MAP
kinase and Rsk did not activate glycogen synthase in either primary (29, 31) or 3T3-L1
adipocytes(28, 32) . MAP kinase activation by insulin
in 3T3-L1 adipocytes was blocked by a novel inhibitor of MEK, PD
098059, without inhibiting the effect of insulin on glycogen
synthase(30) . PHAS-I phosphorylation is increased by agonists
that activate MAP kinase; however, the phosphorylation of PHAS-I by
insulin was not attenuated by inhibition of MAP kinase activation with
PD 098059(22) . In contrast, the effect of insulin on
increasing PHAS-I phosphorylation and promoting the dissociation of the
PHAS-IeIF-4E complex was markedly inhibited by
rapamycin(21, 22) . Rapamycin acts to inhibit
transduction through a pathway that is distinct from the Ras-MAP kinase
pathway and that leads to the activation of
p70
(33, 34, 35, 36) .
Rapamycin was without effect on the activation of glycogen synthase by
insulin in rat adipocytes (29, 31) but markedly
inhibited the activation of synthase in 3T3-L1 adipocytes(37) .
Multiple pathways had been previously shown to exist for the activation
of glycogen synthase by insulin in rat adipocytes(38) .
Different cell types might utilize different transduction pathways to
activate synthase, and it has been suggested that the mechanism of
activation of synthase in skeletal muscle differs from that in
adipocytes.
In view of the importance of skeletal muscle in the regulation of glycogen and protein metabolism by insulin, it is important to determine which signaling pathways are utilized by insulin in this tissue. In the present experiments, we have investigated the roles of the MAP kinase and rapamycin-sensitive pathways in mediating the effects of insulin on glycogen synthase and PHAS-I in rat diaphragm.
For measurements of
glycogen synthase, powdered muscle (100 mg) was homogenized using a
tissue grinder (Teflon-glass) at 4 °C in a solution (1 ml) composed
of 100 mM potassium fluoride, 10 mM EDTA, 2 mM EGTA, 5 mM sodium potassium phosphate, 2 mM potassium phosphate, 50 mM Tris-HCl, pH 7.8, and protease
inhibitors benzamidine (1 mM), leupeptin (10 µg/ml),
aprotinin (10 µg/ml), and phenylmethylsulfonyl fluoride (0.1
mM). Homogenates were centrifuged at 10,000 g for 30 min, and supernatants were retained. When protein kinase
activities were to be measured, powders were homogenized in buffer
containing 10 mM MgCl
, 1 mM sodium
orthovanadate, 1 mM dithiothreitol, 0.1 µM microcystin, 1 mM EDTA, 5 mM EGTA, 10 mM potassium phosphate, 50 mM sodium
-glycerophosphate,
pH 7.4, and the same protease inhibitors as used in the synthase
homogenization buffer. After preparation of extracts by centrifugation
(10,000
g for 30 min), the protein content of each
sample was measured (41) and adjusted to a concentration of 1
mg/ml by adding homogenization buffer.
Rsk activity was
measured by mixing beads with 10 µl of solution containing 50
mM sodium -glycerophosphate (pH 7.4), 14 mM sodium fluoride, 10 mM MgCl
, 1 mM dithiothreitol, 9 µM cAMP-dependent protein kinase
inhibitory peptide(51) , 20 µM calmidazolium, 200
µM [
-
P]ATP (300-500
cpm/pmol), and either 30 µM S6 peptide or 0.1 mg/ml
[His
]
R
(described below). The
mixtures were incubated for 20 min at 30 °C before the reactions
were terminated.
The S6 peptide, KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK,
is based on a phosphorylated region of ribosomal protein S6 and was
used by Dent et al.(9) to assay the activity of the
R kinase, ISPK, later identified as
Rsk-2(10, 11) . With S6 peptide as substrate, the
reactions were terminated by spotting samples (15 µl) onto
phosphocellulose papers (1
2 cm, Whatman P81) and immediately
immersing the papers in 175 mM H
P0
.
The amounts of
P incorporated into the peptide were
determined after washing the papers as described
previously(29) . When [His
]
R
was used as substrate for Rsk, the reactions were terminated by
adding SDS sample buffer. Samples were subjected to
SDS-PAGE(52) , and the relative amounts of
P
incorporated into [His
]
R
were
determined by using a phosphorimager (Molecular Dynamics).
To
measure p70 activities, immune complexes were incubated
as described for measuring Rsk activities, except that 40 S ribosomes
(2 mg/ml final concentration) were used as substrate. The ribosomes
were purified from rat liver as described by Krieg et
al.(53) . The protein kinase reaction was terminated by
adding SDS sample buffer, and samples were subjected to
SDS-PAGE(52) . The relative amounts of
P
incorporated into ribosomal protein S6 were determined by
phosphorimaging.
Figure 1: Effects of insulin and EGF on glycogen synthase activity in rat diaphragm muscles. Diaphragms were incubated at 37 °C without additions (circles) or with 20 nM insulin (squares) or 100 nM EGF (triangles) for increasing times. Glycogen synthase activities were measured in muscle extracts and are expressed as activity ratios. Mean values ± S.E. from four experiments are presented. The inset is an immunoblot showing glycogen synthase (GS) from extracts of muscles that had been incubated for 30 min without additions (CON), with 20 nM insulin (INS) or with 100 nM EGF (EGF).
EGF was much more effective than
insulin in activating MAP kinase. In Fig. 2, the ERK-1 and ERK-2
isoforms of MAP kinase in extracts of insulin- and EGF-treated muscles
were resolved by SDS-PAGE before kinase activities were measured after
renaturation by using an in-gel assay(49) . Bands of activity
corresponding to species of apparent M =
44,000 and 42,000 were detected (Fig. 3A, for example).
These species had exactly the same electrophoretic mobilities as the
ERK-1 and ERK-2 isoforms from adipocytes. (
)There was no
indication of sequential activation (ERK-1 before ERK-2) of the
isoforms as was reported to occur in hindlimb muscles when rats were
injected with insulin(55) . Insulin increased the activities of
ERK-1 (Fig. 2A) and ERK-2 (Fig. 2B) by
only 50-70%. By comparison, EGF increased the activities of the
ERK-1 and ERK-2 by approximately 3- and 5-fold, respectively.
Figure 2: Effects of insulin and EGF on ERK-1 and ERK-2 activities. Diaphragms were incubated as described in Fig. 1. Samples (25 µg of protein) of muscle extracts were subjected to SDS-PAGE in gels containing MBP, and MAP kinase activities were measured after renaturation by using the ``in-gel'' assay described by Wang and Erikson(49) . Relative activities of ERK-1 and -2 were determined by phosphorimaging. Results are expressed as percent of controls, which were activities detected in extracts of cells incubated without insulin or EGF. Mean values ± S.E. from four experiments are presented.
Figure 3:
Inhibition of ERK-1 and ERK-2 activities
by the MEK inhibitor, PD 098059. Diaphragms were incubated for 30 min
at 37 °C without (No Inhibitor) or with 25 µM PD 098059. Incubations were then continued for 30 min without
further additions (No Agonist) or after adding insulin (20
nM) or EGF (100 nM). Samples of extracts were
subjected to SDS-PAGE, and ERK-1 and ERK-2 activities were assessed
after renaturation(49) . A representative autoradiogram is
presented in panel A. Relative activities of ERK-1 (B) and ERK-2 (C) were determined from P
incorporated into MBP. The results are expressed as percentages of
control values and are means ± S.E. from six experiments. ERK-1
activity was also assessed after immunoprecipitation with specific
ERK-1 antibodies (D). In this case, activities are expressed
as pmol of
P incorporated into MBP per mg of extract
protein and are mean values ± S.E. from four
experiments.
The
finding that both isoforms of MAP kinase were markedly increased by EGF
under conditions in which glycogen synthase activity was not changed
indicates that MAP kinase activation is not sufficient for the
activation of glycogen synthase. Because of the important implications
of the findings, control experiments were performed to verify that EGF
was more effective than insulin in activating MAP kinase. EGF produced
a larger increase than insulin when total MAP kinase activity was
measured in nonfractionated extracts or when the activity
of the ERK-1 isoform was measured in immune complex assays (Fig. 3D). Thus, the difference between the
effectiveness of EGF and insulin was not dependent on the method used
to assay MAP kinase. MAP kinase activity was found to be increased in
single muscle fibers that had been manually dissected from control and
EGF-treated muscles,
indicating that the effect of EGF was
not due to effects on other cell types present in skeletal muscle.
Inhibiting MAP kinase activation with the MEK
inhibitor was associated with marked inhibition of Rsk (Fig. 4).
As with MAP kinase activity, Rsk activity measured in an immune complex
assay using a large S6 peptide as substrate was increased much more by
EGF than by insulin. Treating muscles with PD 098059 abolished
activation of Rsk by insulin and decreased the activity observed in the
presence of EGF by approximately 80%. Rsk immunoprecipitated from
muscle extracts readily phosphorylated
[His]
R
, a truncated form of
R
consisting of 443 residues that include site 1 and the
other NH
-terminal sites (Fig. 4B). Both
insulin and EGF increased the activity of Rsk with respect to the
recombinant protein. Incubating muscles with the MEK inhibitor
abolished the effects of both insulin and EGF.
Figure 4:
Attenuation of Rsk activation by MEK
inhibition. Muscles were incubated as described in the legend to Fig. 3. Rsk was immunoprecipitated, and kinase activity was
measured in the immune complexes by using the peptide described by Dent et al.(9) (panel A) or by using
[His]
R
(panel B). The
results are expressed as percentages of the activities observed in
extracts from muscles incubated without additions. Mean values ±
S.E. from three experiments are presented. The inset in panel B is an autoradiogram showing
P-labeled
[His
]
R
phosphorylated by
immunoprecipitated Rsk and [
-
P]ATP. Muscles
were incubated without additions (lane 1) or with 20 nM insulin (lane 2), 100 nM EGF (lane 3),
25 µM PD 098059 (lane 4), PD 098059 plus insulin (lane 5), or PD 098059 plus EGF (lane
6).
Figure 5:
Effects of insulin and EGF on the
association of PHAS-I and eIF-4E in rat skeletal muscle. Diaphragms
were incubated for 30 min at 37 °C without additions (lanes 1 and 4) or with either 20 nM insulin (lanes 2 and 5) or 100 nM EGF (lanes 3 and 6). Samples of extracts (lanes 1-3) or samples
of proteins that had been affinity-purified by using
mGTP-Sepharose (lanes 4-6) were subjected to
SDS-PAGE. An immunoblot showing PHAS-I is presented.
,
,
, and
` are electrophoretic forms of the PHAS-I
protein.
Incubating muscles with insulin for 30 min
dramatically changed the proportions of the different electrophoretic
forms of PHAS-I, indicative of increased phosphorylation of the PHAS-I
protein (Fig. 5). The +
` forms were increased
by approximately 10-fold, and PHAS-I
and
were decreased by
approximately 90 and 75%, respectively. Insulin also markedly decreased
the amount of PHAS-I that copurified with eIF-4E (Fig. 5),
indicating that insulin-stimulated phosphorylation of PHAS-I promotes
the dissociation of the PHAS-I
eIF-4E complex in skeletal muscle.
EGF increased the amount of PHAS-I
`, but the growth factor did
not decrease the amount of PHAS-I bound to eIF-4E. Inhibiting the
activation of MAP kinase with PD 098059 had little if any effect on the
changes in electrophoretic mobility of PHAS-I produced by
insulin.
The results with EGF and PD 098059 indicate that
the regulation of PHAS-I phosphorylation by insulin in skeletal muscle
is mediated by a MAP kinase-independent pathway.
Figure 6:
Effects of insulin and EGF on
p70. Muscles were incubated as described in the legend to Fig. 5. Immunoprecipitations were preformed using antiserum to
p70
(lanes 1, 3, and 5) or
nonimmune serum (lanes 2, 4, and 6), and
samples were subjected to SDS-PAGE. p70
was detected by
immunoblotting. The large band below p70
is IgG
heavy chain (HC), which was derived from the antibodies used
in the immunoprecipitation.
Figure 7:
Rapamycin attenuates the effects of
insulin on p70, glycogen synthase, and PHAS-I. Diaphragms
were incubated at 37 °C for 30 min with increasing concentrations
of rapamycin. Incubations were then continued for 30 min without (circles) or with (squares) 20 nM insulin. A, p70
was immunoprecipitated from extracts, and
kinase activity was measured by using 40 S ribosomes and
[
-
P]ATP as substrates. Activities are
expressed as percentages of maximum kinase activity, which in all
experiments was observed in extracts of muscles that had been incubated
with insulin in the absence of rapamycin. The inset is an
immunoblot showing p70
after immunoprecipitation from
extracts of muscles that had been incubated without additions (CON), with 25 nM rapamycin (RAP), with 20
nM insulin (INS), or with insulin plus rapamycin (INS + RAP). B, glycogen activity ratios were
measured in muscle extracts, and mean values ± S.E. are
presented. Total synthase activities (nmol/min/mg extract protein) were
as follows: control, 33 ± 7; 25 nM rapamycin, 39
± 4; 100 nM rapamycin, 35 ± 4; insulin, 30
± 3; 25 nM rapamycin plus insulin, 34 ± 4; and
100 nM rapamycin plus insulin, 27 ± 6. C,
Extracts were subjected to SDS-PAGE, and the relative amounts of the
and
forms of PHAS-I were determined after immunoblotting
(see inset). The results are expressed as percentages of the
total PHAS-I and are mean values ± S.E. of three experiments. Error bars not shown fall within the symbol.
In the
absence of insulin, rapamycin did not significantly affect glycogen
synthase (Fig. 7B). However, incubating muscles with 25
nM rapamycin attenuated the effect of insulin, and 100 nM rapamycin abolished the effect of the hormone on activating
glycogen synthase (Fig. 7B). Incubating diaphragms with
rapamycin in the absence of insulin also had little effect on the
phosphorylation state of PHAS-I, as the electrophoretic pattern of the
protein was only slightly changed (Fig. 7C). Rapamycin
attenuated, but did not abolish, the effect of insulin on increasing
PHAS-I phosphorylation (Fig. 7C). In muscles incubated
with insulin, rapamycin increased the and
forms by
approximately 4- and 2-fold, respectively.
Activation of MAP kinase by insulin in rat skeletal muscle is neither necessary nor sufficient for the activation of glycogen synthase or the phosphorylation of PHAS-I. Supporting this conclusion are observations that MAP kinase activation by insulin could be abolished by incubating diaphragms with an inhibitor of MEK, which did not significantly attenuate the effects of insulin on activating glycogen synthase (Table 1) or on decreasing PHAS-I binding to eIF-4E (Fig. 5). Moreover, MAP kinase was markedly increased by EGF without activating glycogen synthase (Table 1), and EGF did not mimic the effects of insulin on PHAS-I (Fig. 5). Consequently, it is unlikely that a small amount of insulin-stimulated MAP kinase activity, as might have been undetected in experiments with the MEK inhibitor, could account for the effects of insulin on PHAS-I and glycogen synthase.
The activation of Rsk by insulin was also
abolished by the MEK inhibitor (Fig. 4). This was expected as
Rsk is phosphorylated and activated by MAP
kinase(12, 59) . However, the findings that EGF
activated Rsk without activating glycogen synthase and that inhibiting
Rsk activation with the MEK inhibitor did not abolish synthase
activation by insulin (Fig. 4, Table 1) were not predicted
by the model proposed by Dent et al.(9) in which
synthase activation is mediated by ISPK, the rabbit equivalent of
Rsk(10, 11, 60) . Rsk
immunoprecipitated from diaphragm extracts phosphorylated
[His
]
R
, indicating that the
antibodies recognize forms of the kinase that are capable of
phosphorylating PP1
(Fig. 4). Our interpretation of
the results is that Rsk can phosphorylate R
in
vitro, but Rsk does not mediate the activation of glycogen
synthase by insulin in intact skeletal muscle. It should not be
inferred that synthase activation does not involve phosphorylation of
PP1
, although experiments are needed to confirm that site 1
is phosphorylated in response to insulin in skeletal muscle of rats and
other species in which the hormone activates glycogen synthase.
The
finding that the effects of insulin on activating glycogen synthase and
p70 were inhibited by similar concentrations of rapamycin
is suggestive of a role of the p70
pathway in the control
of synthase (Fig. 7, A and B). Rapamycin is
believed to act by binding FKBP-12, an M
=
12,000 protein that is also the receptor for the immunosuppressant,
FK-506(61, 62) . FKBP-12 possesses peptidyl-prolyl
isomerase activity that is inhibited by both rapamycin and FK506. This
action does not appear to explain the effect of rapamycin to attenuate
insulin signaling, as FK506 blocked the activation of neither
p70
(21, 36) nor glycogen synthase
by insulin. Insensitivity to FK506 would also seem to exclude the
calcium-sensitive phosphatase, calcineurin, in the activation of
synthase, as the FKBP-12
FK506 complex binds to and inhibits
calcineurin(61, 62) . The FKBP-12
rapamycin
complex does not inhibit calcineurin but instead binds to TOR (target
of rapamycin), a protein originally identified in yeast and more
recently cloned from mammalian
cells(63, 64, 65) . The intervening steps
between TOR and p70
have not been identified.
Somewhat
higher concentrations of rapamycin were needed to inhibit synthase or
p70 in the diaphragm than to inhibit p70
in
suspended or cultured
cells(21, 22, 33, 34, 36) ,
where most previous studies of rapamycin sensitivity have been
performed. The possibility that rapamycin acts nonspecifically to
inhibit activation of p70
and glycogen synthase cannot be
excluded. However, to achieve effective concentrations in fibers within
the intact muscle, rapamycin must cross diffusion barriers that are not
present in cultured cells. This may be why higher concentrations of
rapamycin were needed to inhibit p70
in muscle.
Alternatively, TOR or other elements in the signal transduction
pathways leading to the activation of p70
in skeletal
muscle may be less sensitive to rapamycin than those in other cells.
Under conditions in which MAP kinase activity was increased
severalfold by EGF, neither the electrophoretic mobility (Fig. 6) nor the activity of p70
was
significantly changed. Thus, the failure of EGF to activate synthase is
not inconsistent with a role of the p70
pathway in
regulating synthase. One potential mechanism by which activation of
p70
could lead to synthase activation involves GSK-3, the
only protein kinase known to phosphorylate site 3(a+b+c) in
synthase(4, 66) . GSK-3 can be phosphorylated and
inactivated by p70
in vitro(67) , and
insulin has been shown to inhibit GSK-3 activity in
adipocytes(68) , L6 muscle cells(69) , and Chinese
hamster ovary cells overexpressing the human insulin receptor (CHO.T
cells)(70) . Inhibition of GSK-3 alone would be insufficient to
account for dephosphorylation of synthase in rat skeletal muscle, where
insulin promotes dephosphorylation not only of site 3(a+b+c)
but also of site 2(a+b)(5, 71) , which is not
phosphorylated by GSK-3(4, 66) . Moreover, there is
reason to believe that GSK-3 inhibition is not involved in the
rapamycin-sensitive regulation of glycogen synthase, as rapamycin did
not block the effect of insulin on inhibiting GSK-3 activity by insulin
in CHO.T cells (70) or in L6 cells(69) .
The effect of rapamycin on glycogen synthase activity in skeletal muscle is similar to that observed by Shepherd et al.(37) in 3T3-L1 cells but differs from findings in rat adipocytes(29, 31) and CHO cells(72) , where rapamycin did not block the activation of synthase by insulin. Thus, different cells may utilize different pathways for activation of synthase. There is other evidence to support this view. In rat adipocytes glucose potentiates activation of synthase by insulin(38) , whereas in skeletal muscle (5) or 3T3-L1 adipocytes (28) glucose has little if any effect on activating the enzyme. In Swiss mouse 3T3 cells, EGF and platelet-derived growth factor activated glycogen synthase(73, 74) , but EGF did not increase synthase activity in adipocytes (28, 29, 32) or skeletal muscle (Fig. 1). As skeletal muscle is the most important site of insulin-stimulated glycogen deposition(3) , it will be most important to determine the mechanism of synthase activation by insulin in this tissue.
The present study demonstrates
that insulin increases the phosphorylation of PHAS-I and dissociation
of the PHAS-IeIF-4E complex in skeletal muscle. As PHAS-I
inhibits eIF-4E(24) , dissociation would be expected to
increase rates of initiation, providing a potential explanation of the
well established and important action of the hormone on mRNA
translation in skeletal muscle(14) . The phosphorylation of
PHAS-I in response to insulin in skeletal muscle involves a
rapamycin-sensitive pathway (Fig. 7); however, as observed in
adipocytes, rapamycin did not completely inhibit insulin-stimulated
phosphorylation of PHAS-I(21, 22) . Consequently, the
control of phosphorylation of PHAS-I by insulin in skeletal muscle also
involves a rapamycin-insensitive pathway.
The kinase responsible for
the rapamycin-sensitive phosphorylation of PHAS-I has not been
identified. It is unlikely that the PHAS-I kinase is p70,
as purified p70
did not directly phosphorylate
recombinant PHAS-I(27) . This does not exclude the p70
pathway, as kinases either upstream or downstream of p70
might mediate PHAS-I phosphorylation. Similarly, other kinases in
the p70
pathway should be considered as candidates for
mediating the activation of glycogen synthase, perhaps by
phosphorylating site 1 in PP1
. The recent discovery by
Hartley et al.(75) that TOR is homologous to the
DNA-dependent protein kinase catalytic subunit raises the intriguing
possibility that TOR itself may signal as a protein kinase. Although
additional investigation is needed to identify signaling intermediates,
our findings indicate that two of the most important actions of
insulin, the stimulation of both protein and glycogen synthesis in
skeletal muscle, involve a rapamycin-sensitive pathway that is distinct
from the MAP-kinase signaling pathway.