(Received for publication, July 21, 1994; and in revised form, November 1, 1994)
From the
This study was undertaken to define intracellular signaling pathways upstream to glycogen synthase activation. First, we examined the role of the two pathways of insulin signaling, Ras-dependent and wortmannin/LY294002-sensitive, in glycogen synthase activation. Although negative dominant Ras (Ras17N) induction in PC12 cells markedly decreased activities of mitogen-activated protein kinase (MAP) and pp90 S6 kinase in response to insulin or insulin-like growth factor I (IGF-I), activation of glycogen synthase by these agents was unaffected by negative dominant Ras induction. In contrast, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), inhibitors of phosphatidylinositol 3-kinase, antagonized glycogen synthase activation in response to insulin or IGF-I. Next, we examined the contribution of pp70 S6 kinase, one of the wortmannin/LY294002-sensitive signaling molecules on glycogen synthase activation. Immunosuppressant rapamycin completely blocked activation of pp70 S6 kinase by insulin or IGF-I, but rapamycin alone or in combination with induction of negative dominant Ras failed to antagonize glycogen synthase activation by these hormones. These data suggest that 1) activation of Ras-MAP kinase is not necessary for stimulation of glycogen synthase and 2) activation of wortmannin/LY294002sensitive pathway, independent of pp70 S6 kinase, plays a key role in glycogen synthase regulation in PC12 cells.
One of the major physiological roles of insulin is to regulate
fuel metabolism. Insulin stimulates a number of processes that are
central to the deposition of glucose into glycogen. Of these processes,
activation of glycogen synthase is important in insulin's action.
Defects of activating glycogen synthase by this hormone were observed
in insulin resistance of type II diabetes mellitus, implicating defects
in signaling upstream to the
enzyme(1, 2, 3) . The stimulation of glycogen
synthase by insulin and other polypeptide growth factors seems to
possess a common intermediate element in the signaling pathways (4) and results in the dephosphorylation and activation of
glycogen synthase. The precise mechanism by which insulin mediates
signals to activate glycogen synthase, however, is still incompletely
understood. Activation of tyrosine kinase of the insulin receptor might
be the initial step for activating glycogen
synthase(5, 6) , and this signal finally either
activates protein phosphatase-1 (7) by increasing
phosphorylation of site 1 of glycogen-associated subunit (G subunit) of
protein phosphatase-1 (8) or inhibits glycogen synthase
kinase-3 (9, 10) through the yet unidentified
mechanisms. An insulin-stimulated protein kinase, a mammalian homologue
of pp90 S6 kinase (10) has been a candidate molecule to link
between the insulin receptor tyrosine kinase and dephosphorylation of
glycogen synthase because it is shown to phosphorylate and activate the
G subunit of phosphatase-1 and to phosphorylate and inactivate glycogen
synthase kinase-3 in vitro (8, 12). ()These data
suggested that cascade involving insulin-stimulated protein kinase,
originating from p21
(Ras) (
)signaling pathway leading to activation of
mitogen-activated protein (MAP) kinase and pp90 S6 kinase, might play a
pivotal role in glycogen synthesis.
In addition to the Ras-MAP kinase cascade, polypeptide growth factors, including insulin, activate PI 3-kinase cascade(13) . In insulin-sensitive cell lines, most of the PI 3-kinase activity is associated with insulin receptor substrate-1 by the interaction of the Src homology 2 domains of the 85-kDa subunit of PI 3-kinase with the YXXM motifs of insulin receptor substrate-1(14, 15) . Recently, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), inhibitors for PI 3-kinase(16, 17) , were reported to inhibit stimulation of 2-deoxyglucose uptake and inhibition of isoproterenol-induced lipolysis by insulin(16, 18) , suggesting that wortmannin/LY294002-sensitive mechanisms might lie upstream to these metabolic responses. These observations inspired us to examine whether wortmannin/LY294002-sensitive mechanism would contribute to glycogen synthase activation, another important metabolic response of insulin. Downstream to wortmannin/LY294002-sensitive signaling pathway lies pp70 S6 kinase(18, 19, 20) . pp70 S6 kinase phosphorylated and inactivated glycogen synthase kinase-3 in vitro(12) , raising the possibility that pp70 S6 kinase could contribute to stimulation of glycogen synthesis.
Prior studies implicating insulin-sensitive protein kinase and pp70 S6 kinase in glycogen synthase activation have relied exclusively on in vitro systems, and there were no reports examining the contribution of wortmannin/LY294002-sensitive pathway on insulin-induced glycogen synthase activation to date. Thus, we have investigated the role of Ras and wortmannin/LY294002-sensitive pathway, including pp70 S6 kinase, in the insulin-induced activation of glycogen synthase in PC12 cells. PC12 cells (21) were reported to possess receptors for insulin and IGF-I(22) , to stimulate the conversion of the inactive GDP-bound form of Ras to the active GTP-bound form(23) , to stimulate S6 kinase activity(24) , and to stimulate glycogen synthase (25) in response to insulin or IGF-I. In the present study, we found that glycogen synthase activation evoked by insulin or IGF-I was not affected by inhibiting Ras activation by induction of negative dominant Ras (Ras17N) but significantly affected by treatment with wortmannin or LY294002, indicating that glycogen synthase activation by insulin or IGF-I in PC12 cells is largely under the control of wortmannin/LY294002-sensitive pathway, not downstream to Ras-MAP kinase cascade. We also observed that treatment with rapamycin, alone or in combination with negative dominant Ras, failed to antagonize glycogen synthase activation by these hormones, indicating glycogen synthase activation was not downstream to pp70 S6 kinase.
Figure 1:
Effects of Ras17N
induction on MAP kinase, pp90 S6 kinase, pp70 S6 kinase, and glycogen
synthase. PanelA, PC12 cells were pretreated with
low serum medium containing 1 µM dexamethasone for 24 h
and stimulated with 1 µM insulin or 100 nM IGF-I.
Immune complex kinase assays were performed as described under
``Experimental Procedures.'' The results were converted to
the ratio against the unstimulated figure of PC12-DEX cells, and are
the means of three separate experiments shown in triplicate (means
± S.D.). PanelB, PC12 cells were pretreated
with low serum medium containing 1 µM dexamethasone for 24
h and stimulated with various concentrations of IGF-I. Glycogen
synthase activity was measured as described under ``Experimental
Procedures.'' The results (in dpm) were converted to the activity
ratios determined by dividing the activity measured with 0.25 mM glucose 6-phosphate by the activity measured with 10 mM glucose 6-phosphate and are the means of three separate
experiments shown in duplicate (means ± S.D.). , PC12-DEX;
, PC12Ras17N-DEX. PanelC, PC12 cells were
pretreated with low serum medium containing 1 µM dexamethasone for 24 h and stimulated with various concentrations
of insulin. Glycogen synthase activity was measured as described under
``Experimental Procedures.'' The results (in dpm) were
converted to the activity ratios and are the means of three separate
experiments shown in duplicate (means ± S.D.).
, PC12-DEX;
, PC12Ras17N-DEX.
Figure 2:
Effects of wortmannin on PI 3-kinase, MAP
kinase, pp90 S6 kinase, pp70 S6 kinase, and glycogen synthase. PanelA, PI 3-kinase activity in PY
immunoprecipitates from cells pretreated without (vehicle, 0.01%
dimethyl sulfoxide) or with various concentrations of wortmannin and
stimulated with or without IGF-I was analyzed as described under
``Experimental Procedures.'' Autoradiograms were exposed at
-70 °C for 12 h. PanelB, PC12 cells were
preincubated for 30 min without (vehicle, 0.01% dimethyl sulfoxide) or
with various concentrations of wortmannin and incubated for a further
45 min with or without 100 nM IGF-I. Glycogen synthase
activity was measured as described under ``Experimental
Procedures.'' The results are expressed as the (means ±
S.D.) percentages of maximal stimulation by IGF-I and are the means of
three separate experiments in duplicate. PanelC,
PC12 cells were preincubated for 30 min without (vehicle, 0.01%
dimethyl sulfoxide) or with various concentrations of wortmannin and
incubated for a further 30 min with or without 100 nM IGF-I.
pp70 S6 kinase activity was measured as described under
``Experimental Procedures.'' The results are expressed as the
(means ± S.D.) percentages of maximal stimulation by IGF-I and
are the means of two separate experiments in triplicate. PanelD, PC12 cells were pretreated with low serum medium for
24 h, preincubated for 30 min without (vehicle, 0.01% dimethyl
sulfoxide) or with 30 nM wortmannin, and incubated with 1
µM insulin or 100 nM IGF-I for a further 5 min
(MAP kinase) or 10 min (pp90 S6 kinase). Immune complex kinase assays
were performed as described under ``Experimental
Procedures.'' The results were converted to the ratio against the
unstimulated figure of PC12 cells and are the means of two separate
experiments shown in triplicate (means ±
S.D.).
Figure 3:
Effects of LY294002 on PI 3-kinase, MAP
kinase, pp90 S6 kinase, pp70 S6 kinase, and glycogen synthase. PanelA, PI 3-kinase activity in PY
immunoprecipitates from cells pretreated without (vehicle, 1% dimethyl
sulfoxide) or with various concentrations of LY294002 and stimulated
with or without IGF-I was analyzed as described under
``Experimental Procedures.'' Autoradiograms were exposed at
-70 °C for 12 h. PanelB, PC12 cells were
preincubated for 5 min without (vehicle, 1% dimethyl sulfoxide) or with
various concentrations of LY294002 and incubated for a further 45 min
with or without 100 nM IGF-I. Glycogen synthase activity was
measured as described under ``Experimental Procedures.'' The
results are expressed as the (means ± S.D.) percentages of
maximal stimulation by IGF-I and are the means of three separate
experiments in duplicate. PanelC, PC12 cells were
preincubated for 5 min without (vehicle, 1% dimethyl sulfoxide) or with
various concentrations of LY294002 and incubated for a further 30 min
with or without 100 nM IGF-I. pp70 S6 kinase activity was
measured as described under ``Experimental Procedures.'' The
results are expressed as the (means ± S.D.) percentages of
maximal stimulation by IGF-I and are the means of two separate
experiments in triplicate. PanelD, PC12 cells were
pretreated with low serum medium for 24 h, preincubated for 5 min
without (vehicle, 1% dimethyl sulfoxide) or with 30 µM LY294002, and incubated for a further 5 min (MAP kinase) or 10 min
(pp90 S6 kinase) with 1 µM insulin or 100 nM IGF-I. Immune complex kinase assays were performed as described
under `` Experimental Procedures.'' The results were
converted to the ratio against the unstimulated figure of PC12 cells
and the means of two separate experiments in triplicate (means ±
S.D.).
Figure 4:
Effects of rapamycin on MAP kinase, pp90
S6 kinase, pp70 S6 kinase, and glycogen synthase. PanelA, PC12 cells were pretreated with low serum medium for
24 h, preincubated for 30 min without (vehicle, 0.25% ethanol) or with
25 ng/ml rapamycin, and incubated for a further 5 min (MAP kinase), 10
min (pp90 S6 kinase), or 30 min (pp70 S6 kinase) with 1 µM insulin or 100 nM IGF-I. Immune complex kinase assays
were performed as described under ``Experimental
Procedures.'' The results were converted to the ratio against the
unstimulated figure of PC12 cells and are the means of two separate
experiments in triplicate (means ± S.D.). PanelB, PC12 cells were preincubated for 30 min without
(vehicle, 0.25% ethanol) or with 25 ng/ml rapamycin and incubated for
further a 45 min with various concentrations of IGF-I. Glycogen
synthase activity was measured as described under ``Experimental
Procedures.'' The results (in dpm) were converted to the activity
ratios determined by dividing the activity measured with 0.25 mM glucose 6-phosphate by the activity measured with 10 mM glucose 6-phosphate and are the means of three separate
experiments in duplicate (means ± S.D.). , with rapamycin;
, without rapamycin. PanelC, PC12Ras17N cells
were pretreated with low serum medium containing 1 µM dexamethasone for 24 h, preincubated for 30 min without (vehicle,
0.25% ethanol) or with 25 µg/ml rapamycin, and incubated for a
further 45 min with various concentrations of IGF-I. Glycogen synthase
activity was measured as described under ``Experimental
Procedures.'' The results (in dpm) were converted to the activity
ratios and are the means of three separate experiments in duplicate
(means ± S.D.).
, with rapamycin;
, without
rapamycin.
The defect in insulin action observed in type II diabetic patients affects insulin-stimulated glycogen synthesis as well as glucose uptake (1, 2) , suggesting defects in signaling upstream to glycogen synthase. The longstanding paradox that insulin promotes the phosphorylation of some proteins while this hormone also promotes the dephosphorylation of glycogen synthase was recently explained that the latter mechanism was due to activation of the G subunit of type 1 protein phosphatase and inactivation of glycogen synthase kinase-3 by phosphorylation(8, 12) . To date, pp90 S6 kinase and/or pp70 S6 kinase have been believed to be involved in the process because pp90 S6 kinase phosphorylated both G subunits of protein phosphatase-1 and glycogen synthase kinase-3 and pp70 S6 kinase phosphorylated glycogen synthase kinase-3 in vitro(8, 12) . In addition, wortmannin and LY294002 were recently reported to inhibit stimulation of 2-deoxyglucose uptake(16, 18) , raising the possibility that wortmannin/LY294002-sensitive mechanisms might lie upstream to metabolic responses of insulin in common. Thus, the present experiments were designed to investigate whether the upstream mechanism of glycogen synthase activation would be Ras-MAP kinase-dependent or wortmannin/LY294002-sensitive and to evaluate the hypothesis that activation of pp90 S6 kinase and/or pp70 S6 kinase mediates glycogen synthase activation in intact cell system.
Several
insulin-stimulated signal transduction pathways have been shown to be
mediated by Ras. Insulin, as well as other polypeptide growth factors,
activates Ras by stimulating the conversion of the inactive GDP-bound
form of Ras to the active GTP-bound form(35, 36) . Ras
activates the downstream kinases (37, 38, 39) such as MAP kinase and pp90 S6
kinase (or an insulin-sensitive protein
kinase)(28, 40, 41) , and the latter is
presumably involved in glycogen synthesis. As shown in Fig. 1,
we found that inhibition of Ras by Ras17N did not affect glycogen
synthase activity stimulated by insulin or IGF-I, although the
activation of MAP kinase and pp90 S6 kinase and cellular growth by
these hormones were impaired. This observation in vivo differed from those observed in vitro(8, 11) and provides first direct evidence that
activation of Ras, MAP kinases, and pp90 S6 kinase is not required to
activate glycogen synthase in response to insulin or IGF-I. We also
observed no significant increase in glycogen synthase activity in
response to insulin in Chinese hamster ovary cells overexpressing human
insulin receptor and MAP kinase, although in these cell lines, pp90 S6
kinase activity was more sensitive to insulin than the parental cell
lines overexpressing human insulin receptor. ()These data,
together with the recent reports that epidermal growth factor activates
Ras signaling but fails to activate glycogen synthase in 3T3-L1
adipocytes and rat adipocytes(42, 43) , strongly
suggest that activation of Ras-MAP kinase pathway is neither necessary
nor sufficient for activation of glycogen synthase by polypeptide
growth factors in these cell lines.
Wortmannin/LY294002-sensitive
pathway is another important cascade in insulin signaling(13) .
PI 3-kinase was initially characterized as a phosphatidylinositol
kinase that associated with activated tyrosine kinases or
tyrosine-phosphorylated proteins such as
IRS-I(14, 15) . Recently, distinct forms of PI
3-kinase activated by subunits of GTP-binding proteins (44) as well as mammalian target of rapamycin (also called
RAFT1 and FRAP) with strong sequence similarity to the lipid kinase
domain of p110
and
of PI 3-kinase (45, 46, 47) were characterized. These
``PI 3-kinase family'' molecules also seem to be targets of
LY294002, inhibitors originally designed as specific inhibitors of PI
3-kinase associated with activated tyrosine kinase (25) , and
low concentrations of wortmannin(16, 44) . Wortmannin
and/or LY294002 antagonized insulin-induced stimulation of
2-deoxyglucose uptake(16, 18) , inhibition of
isoproterenol-induced lipolysis by insulin(18) , and activation
of pp70 S6 kinase(18, 19, 20) . As molecules
of the PI 3-kinase family responsible for activating the above
processes have not yet been specified, these processes are now defined
as wortmannin/LY294002-sensitive(48) . It is interesting that
activation of glycogen synthase by insulin or IGF-I was strongly
inhibited by low concentrations of wortmannin (Fig. 2B). Under these conditions, wortmannin did not
affect MAP kinase nor pp90 S6 kinase activities significantly (Fig. 2D). In addition, the inhibitory effect of
LY294002 was observed on glycogen synthase activation with an IC
of 13 µM (Fig. 3B). This value is
comparable with that to prevent proliferation of smooth muscle cells in
cultured rabbit aortic segments (IC
, 32
µM(17) ). These observations have strongly
suggested that the wortmannin/LY294002-sensitive pathway might be a
point of divergence in stimulating glycogen synthesis in PC12 cells.
pp70 S6 kinase is another kinase that phosphorylates glycogen synthase kinase-3 in vitro(12) . This kinase is known to be activated by polypeptide growth factors including insulin(49, 50) , the upstream mechanism of which is independent on Ras-signaling cascades (18) (Fig. 1A) and dependent on wortmannin/LY294002-sensitive mechanisms(18, 19, 20) . In the present study, rapamycin failed to antagonize glycogen synthase activation by insulin or IGF-I, although pp70 S6 kinase activity was abolished by rapamycin treatment. In addition, inhibitory profiles of LY294002 on glycogen synthase activation was somewhat different from those of pp70 S6 kinase activation, supporting the conclusion drawn from experiments on rapamycin treatment. Thus, we demonstrated that pp70 S6 kinase is not involved in the activation of glycogen synthase by these hormones in PC12 cells. Similar results were recently reported in rat adipocytes that epidermal growth factor activates pp70 S6 kinase but fails to activate glycogen synthase and that activation of glycogen synthase was not antagonized by rapamycin(43) . Moreover, it is also unlikely that pp90 S6 kinase and pp70 S6 kinase are complementary; that is, activation of either S6 kinase is sufficient for glycogen synthase activation because rapamycin, in combination with Ras17N induction, also failed to antagonize glycogen synthase activation by these hormones (Fig. 2C).
In summary, we propose that wortmannin/LY294002-sensitive mechanism, not pp70 S6 kinase nor Ras-MAP kinase-signaling cascade, might be a point of divergence in glycogen synthase activation in PC12 cells. This novel finding might provide a clue to pathogenesis of impaired activation of glycogen synthase observed in type II diabetes mellitus.