A Rapamycin-Sensitive Pathway Down-Regulates Insulin Signaling via Phosphorylation and Proteasomal Degradation of Insulin Receptor Substrate-1
Tetsuro Haruta,
Tatsuhito Uno,
Junko Kawahara,
Atsuko Takano,
Katsuya Egawa,
Prem M. Sharma,
Jerrold M. Olefsky and
Masashi Kobayashi
First Department of Medicine (T.H., T.U., J.K., A.T., M.K.)
Toyama Medical and Pharmaceutical University Toyama, 930-0194,
Japan
Department of Medicine (K.E., P.M.S., J.M.O.)
Division of Endocrinology and Metabolism and Whittier Diabetes
Institute University of California, San Diego La Jolla,
California 92093
Veterans Administration Research Service
(J.M.O.) San Diego, California 92161
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ABSTRACT
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Insulin receptor substrate-1 (IRS-1) is a major
substrate of the insulin receptor and acts as a docking protein for Src
homology 2 domain containing signaling molecules that mediate many of
the pleiotropic actions of insulin. Insulin stimulation elicits
serine/threonine phosphorylation of IRS-1, which produces a mobility
shift on SDS-PAGE, followed by degradation of IRS-1 after prolonged
stimulation. We investigated the molecular mechanisms and the
functional consequences of these phenomena in 3T3-L1 adipocytes. PI
3-kinase inhibitors or rapamycin, but not the MEK
inhibitor, blocked both the insulin-induced electrophoretic mobility
shift and degradation of IRS-1. Adenovirus- mediated expression of a
membrane-targeted form of the p110 subunit of phosphatidylinositol (PI)
3-kinase (p110CAAX) induced a mobility shift
and degradation of IRS-1, both of which were inhibited by rapamycin.
Lactacystin, a specific proteasome inhibitor, inhibited insulin-induced
degradation of IRS-1 without any effect on its electrophoretic
mobility. Inhibition of the mobility shift did not significantly affect
tyrosine phosphorylation of IRS-1 or downstream insulin signaling. In
contrast, blockade of IRS-1 degradation resulted in sustained
activation of Akt, p70 S6 kinase, and mitogen-activated protein (MAP)
kinase during prolonged insulin treatment. These results indicate that
insulin-induced serine/threonine phosphorylation and degradation of
IRS-1 are mediated by a rapamycin-sensitive pathway, which is
downstream of PI 3-kinase and independent of ras/MAP kinase. The
pathway leads to degradation of IRS-1 by the proteasome, which plays a
major role in down-regulation of certain insulin actions during
prolonged stimulation.
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INTRODUCTION
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The insulin receptor mediates insulin action through tyrosine
phosphorylation of endogenous substrates, which can recruit Src
homology 2 (SH2) domain containing signaling molecules such as the
p85-regulatory subunit of phosphatidylinositol (PI) 3-kinase, Grb2,
Nck, Crk, Fyn, SHP-2, and others (1, 2). The PI 3- kinase pathway
and the ras/mitogen-activated protein (MAP) kinase pathway represent
two major elements of insulin receptor signaling, which include
multiple protein serine/threonine (Ser/Thr) kinases (1, 2). One of the
most important actions of insulin is to enhance incorporation of
nutrients into cells. For example, insulin stimulates glucose
incorporation into target cells such as skeletal muscle, heart muscle,
and adipose cells (1, 2). System A amino acid transport, glycogen
synthesis, antilypolysis, and inhibition of gluconeogenesis are also
stimulated by insulin (3, 4, 5, 6, 7, 8). Insulin also promotes protein synthesis,
in which the mammalian target of rapamycin (mTOR), also termed
FKBP-rapamycin-associated protein (FRAP) or rapamycin and FKBP12
targets 1 (RAFT1), plays an important role (9). mTOR controls two
translational components, eIF-4E binding protein 1 (4E-BP1) and p70 S6
kinase. Phosphorylation of 4E-BP1 upon stimulation releases eukaryotic
initiation factor 4E (eIF-4E), allowing it to form a productive
initiation complex (9). p70 S6 kinase functions to increase translation
of 5'- terminal oligopyrimidine tract mRNAs, which largely code for
ribosomal proteins and other elements of the translational machinery
(9).
Insulin receptor substrate-1 (IRS-1) is an endogenous substrate of the
insulin receptor and contains at least 20 potential tyrosine
phosphorylation sites and more than 30 potential Ser/Thr
phosphorylation sites (10). Even in the basal state, IRS-1 is highly
phosphorylated on Ser and Thr residues, which explains the migration of
IRS-1 on SDS-PAGE at an electrophoretic mobility corresponding to
approximately 185 kDa, compared with its predicted molecular mass of
131 kDa (10). Insulin stimulation immediately elicits IRS-1
phosphorylation on both tyrosine and Ser/Thr residues (11) and
subsequently produces a shift in mobility on SDS-PAGE due to further
Ser/Thr phosphorylation (11).
Recent studies in cultured cells, animal models, and human disease
states have suggested that Ser/Thr phosphorylation of IRS-1 may be
involved in the development of insulin resistance (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25). Several
studies have attempted to identify a Ser/Thr kinase responsible for
phosphorylation of IRS-1 that leads to attenuation of insulin
signaling. For example, glycogen synthase kinase-3 was reported to
phosphorylate IRS-1 and inhibit insulin receptor tyrosine kinase
activity (20). Activation of protein kinase C by phorbol
12-myristate-13-acetate elicits Ser phosphorylation of IRS-1, which is
mediated by MAP kinase and leads to a decreased ability of the insulin
receptor to tyrosine-phosphorylate IRS-1 with decreased p85 association
(19). Platelet-derived growth factor (PDGF) stimulation causes an IRS-1
mobility shift due to Ser/Thr phosphorylation, and this effect is
mediated through PI 3-kinase (23, 24). More recently, Akt (also called
protein kinase B) was found to mediate Ser/Thr phosphorylation of
IRS-1, and this was inhibited by rapamycin (25). However, the molecular
mechanisms and the functional consequences of insulin-induced Ser/Thr
phosphorylation of IRS-1 are largely unknown.
Furthermore, earlier studies indicated that chronic exposure of cells
to insulin resulted in degradation of IRS-1 protein (26, 27, 28, 29, 30). The rate
of IRS-1 degradation was about 10 times faster in insulin-treated cells
than in basal cells (26). Although a role for PI 3-kinase in
insulin-induced degradation of IRS-1 was suggested (28), little is
known about the mechanism and the role of insulin-induced degradation
of IRS-1, and the relationship between phosphorylation and degradation
of IRS-1 has not been investigated.
In the present study, we have explored the molecular mechanisms of
insulin-stimulated Ser/Thr phosphorylation and degradation of IRS-1, as
well as the functional consequences. We show here that a
rapamycin-sensitive pathway is involved in both of these phenomena,
which eventually mediates degradation of IRS-1 by the proteasome. Our
data suggest a role for this pathway in down-regulation of insulin
action during prolonged stimulation.
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RESULTS
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Insulin Induces an Electrophoretic Mobility Shift and Degradation
of IRS-1
To investigate the molecular mechanism of the insulin-induced
mobility shift and degradation of IRS-1, we first determined the time
course of these effects, as well as tyrosine phosphorylation of IRS-1
and the phosphorylation status of molecules that mediate insulin
action. Fully differentiated 3T3-L1 adipocytes were stimulated with 20
nM insulin for various periods, and cell lysates were
subjected to SDS-PAGE followed by immunoblotting with anti-IRS-1,
phosphotyrosine, phospho-specific Akt, phospho-specific p70 S6 kinase,
or phospho-specific MAP kinase antibodies (Fig. 1
). As reported previously (11), insulin
stimulation increased the apparent molecular mass of IRS-1 in a
time-dependent manner with the maximal gel mobility shift observed at
1 h after stimulation (Fig. 1
). When the cells were stimulated for
longer time periods, the mobility of IRS-1 gradually returned to the
basal state, and the intensity of the IRS-1 band decreased in a
time-dependent manner, indicating that IRS-1 protein was gradually
degraded, as previously shown (26, 27, 28, 29, 30). IRS-1 protein levels decreased
to 31% and 12% of the original level at 4 and 8 h after insulin
stimulation, respectively. Even at 1 h after insulin stimulation,
IRS-1 protein level was decreased to 75% (Fig. 1
). At 24 h after
stimulation with insulin, the IRS-1 protein level was somewhat
recovered compared with that at 8 h after insulin stimulation
(Fig. 1
). Tyrosine phosphorylation of pp185, most of which is IRS-1 in
3T3-L1 adipocytes (31), was induced rapidly in response to insulin,
with a maximum at 15 min after insulin stimulation, a gradual
decrease thereafter, and a slight recovery at 24 h (Fig. 1
). Thus,
the time course of pp185 tyrosine phosphorylation apparently correlated
with IRS-1 protein levels. However, densitometric analysis showed that
pp185 levels decreased only to 57% and 37% of the original level at 4
and 8 h after insulin stimulation, respectively (Fig. 1
),
indicating that the decrease of pp185 levels is less than that of IRS-1
protein levels. The difference may be due to a continuous increase in
tyrosine phosphorylation of IRS-1 and/or preferential degradation of
IRS-1 molecules that are not phosphorylated on tyrosine residues.
Phosphorylation of Akt on Ser473 was rapidly induced at 1 min after
stimulation with insulin, remained stable for 2060 min, and then
gradually decreased (Fig. 1
). A similar time course was observed for
phosphorylation of Thr308 of Akt (Fig. 1
). Phosphorylation of p70 S6
kinase on Ser411 was induced more slowly, reaching a maximum at 2060
min after insulin stimulation, decreasing slowly thereafter (Fig. 1
).
Phosphorylation of Thr389 of p70 S6 kinase showed a similar time course
(Fig. 1
). Phosphorylation of MAP kinase was rapidly stimulated and was
maximal at 5 min after insulin stimulation and gradually decreased
thereafter (Fig. 1
).

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Figure 1. Time Course of Electrophoretic Mobility Shift and
Degradation of IRS-1
Fully differentiated 3T3-L1 adipocytes were serum starved for 16 h
and stimulated with 20 nM insulin for indicated periods of
time. Cell lysates were then analyzed by electrophoresis and
immunoblotting with anti-IRS-1, antiphosphotyrosine, antiphospho-Ser473
Akt, antiphospho-Thr308 Akt, antiphospho-Ser411 p70 S6 kinase,
antiphospho-Thr389 p70 S6 kinase, or antiphospho-Thr202/Tyr204 MAP
kinase antibody. Protein amounts of Akt, p70 S6 kinase, and MAP kinase
as assessed by immunoblotting with non-phospho-specific antibodies were
not affected during the period examined (data not shown). Data were
analyzed by densitometry and means ± SE of three
independent experiments are shown (lower panels).
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PI 3-Kinase Inhibitors and Rapamycin, but Not the MEK Inhibitor
Block the Insulin-Induced Mobility Shift of IRS-1
To determine which insulin signaling pathway mediates the
insulin-induced mobility shift of IRS-1, we examined the effects of PI
3-kinase inhibitors, rapamycin (which inhibits mTOR signaling pathway),
or the MEK inhibitor. Since maximal mobility shift was observed at 60
min after insulin stimulation (Fig. 1
), cells were stimulated with
insulin for 60 min after pretreatment with various concentrations of
each inhibitor. The mobility of IRS-1 and the phosphorylation status of
downstream kinases were monitored by immunoblot analysis of cell
lysates. Pretreatment of the cells with a PI 3-kinase inhibitor,
wortmannin, inhibited the insulin-induced mobility shift of IRS-1 in a
dose- dependent manner, along with a dose-dependent inhibition of
phosphorylation of both Akt and p70 S6 kinase (Fig. 2A
). A structurally different PI 3-kinase
inhibitor, LY294002, had similar effects (Fig. 2B
). Pretreatment of the
cells with rapamycin also resulted in a dose-dependent inhibition of
the insulin-induced mobility shift of IRS-1 (Fig. 2C
), along with a
dose-dependent inhibition of phosphorylation of p70 S6 kinase, but not
phosphorylation of Akt (Fig. 2C
). The MEK inhibitor, PD98059, had no
significant effect on the IRS-1 mobility or phosphorylation of Akt, and
only a slight effect on phosphorylation of p70 S6 kinase, while it
inhibited insulin-induced MAP kinase phosphorylation in a
dose-dependent manner (Fig. 2D
).

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Figure 2. Effects of PI 3-Kinase Inhibitors, Rapamycin or
PD98059, on Insulin-Induced Electrophoretic Mobility Shift of IRS-1
Fully differentiated 3T3-L1 adipocytes were serum starved for 16 h
and pretreated with indicated concentrations of wortmannin (panel A),
LY294002 (panel B), rapamycin (panel C), PD98059 (panel D) or vehicle
[dimethylsulfoxide (DMSO), final 0.1%] for 30 min before stimulation
without or with 20 nM insulin for 60 min. Cell lysates were
then analyzed by electrophoresis and immunoblotting with anti-IRS-1,
antiphospho-Ser473 Akt, antiphospho-Ser411 p70 S6 kinase, or
antiphospho-Thr202/Tyr204 MAP kinase antibody. None of the treatments
affected protein amounts of Akt, p70 S6 kinase, or MAP kinase as
assessed by immunoblotting with non-phospho-specific antibodies (data
not shown). A representative of three independent experiments is shown.
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PI 3-Kinase Inhibitors and Rapamycin, but Not the MEK Inhibitor
Prevent Insulin-Induced Degradation of IRS-1
It has been previously reported that insulin-induced IRS-1
degradation is mediated by a PI 3-kinase- dependent pathway (28).
Therefore, to evaluate this, we examined the effects of the inhibitors
on insulin- induced degradation of IRS-1 and tyrosine
phosphorylation of pp185. Wortmannin almost completely inhibited
insulin-induced degradation of IRS-1 at 4 h after insulin
stimulation at the same concentration that inhibited phosphorylation of
Akt and p70 S6 kinase (Fig. 3A
). The
insulin-induced mobility shift (at 1 h) was also inhibited (Fig. 3A
). The decrease in tyrosine phosphorylation of pp185 was also
inhibited in parallel (Fig. 3A
). Another PI 3-kinase inhibitor,
LY294002, had similar effects (Fig. 3B
). Pretreatment of the cells with
rapamycin, at a concentration that inhibited the insulin-induced
mobility shift, and phosphorylation of p70 S6 kinase also clearly
inhibited IRS-1 degradation at 4 h after insulin stimulation (Fig. 3C
). Tyrosine phosphorylation of IRS-1 again appeared to correlate with
IRS-1 protein levels (Fig. 3C
). PD98059 had no effect on degradation or
tyrosine phosphorylation of IRS-1 (Fig. 3D
). Taken together, the above
results indicate that a rapamycin-sensitive pathway, which is
downstream of PI 3-kinase, but not the ras/MAP kinase pathway, is
required for both insulin-induced Ser/Thr phosphorylation and
degradation of IRS-1.

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Figure 3. Effects of PI 3-Kinase Inhibitors, Rapamycin or
PD98059, on Insulin-Induced Degradation and Tyrosine Phosphorylation of
pp185
Fully differentiated 3T3-L1 adipocytes were serum starved for
16 h and pretreated with indicated concentrations of wortmannin
(panel A), LY294002 (panel B), rapamycin (panel C), PD98059 (panel D),
or vehicle (DMSO, final 0.1%) for 30 min before stimulation without or
with 20 nM insulin for 1 or 4 h. Cell lysates were
then analyzed by electrophoresis and immunoblotting with anti-IRS-1,
antiphosphotyrosine, antiphospho-Ser473 Akt, or antiphospho-Ser411 p70
S6 kinase antibody. None of the treatments affected protein amounts of
Akt, p70 S6 kinase, or MAP kinase as assessed by immunoblotting with
non-phospho-specific antibodies (data not shown). Data were analyzed by
densitometry and means ± SE of three independent
experiments are shown (right panels).
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Overexpression of p110CAAX Induces a
Mobility Shift and Degradation of IRS-1 via a Rapamycin-Sensitive
Pathway
Fully differentiated 3T3-L1 adipocytes were infected with the
adenovirus vector, Ad5-p110CAAX, which expresses
a membrane-targeted form of the catalytic subunit of PI 3-kinase. At
48 h after infection, cell lysates were subjected to SDS-PAGE and
immunoblot analysis. Ad5-p110CAAX caused
phosphorylation of both Akt and p70 S6 kinase (Fig. 4A
), indicating that membrane targeting
of the p110 subunit led to activation of these downstream effector
molecules of PI 3-kinase, as shown previously (32).
Ad5-p110CAAX induced the mobility shift of IRS-1
(Fig. 4A
), although the extent of this was less than that induced by
insulin (data not shown). Since the insulin-induced mobility shift
declines after 1 h (Fig. 1
), the relatively small mobility shift
induced by p110CAAX may be due to continuous
activation of PI 3-kinase. The results also show that
p110CAAX caused a greater loss of IRS-1 protein
(Fig. 4A
), indicating that IRS-1 was degraded as a result of
overexpression of p110CAAX. We examined the
effect of rapamycin on the p110CAAX-induced IRS-1
mobility shift and degradation. As can be seen in Fig. 4B
, the mobility
shift of IRS-1 induced by p110CAAX was inhibited
by rapamycin in a dose- dependent manner. The loss of IRS-1 protein
was also partially restored by rapamycin (Fig. 4B
). Rapamycin also
inhibited the p110CAAX-induced phosphorylation of
p70 S6 kinase in a dose-dependent manner without any effect on Akt
phosphorylation (Fig. 4B
). MAP kinase phosphorylation was not
stimulated by p110CAAX overexpression and was not
affected by rapamycin (Fig. 4B
).

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Figure 4. Effects of p110CAAX and Rapamycin on
Electrophoretic Mobility and Protein Amounts of IRS-1
Fully differentiated 3T3-L1 adipocytes were infected with either Ad5-CT
or Ad5-p110CAAX at the indicated multiplicity of infection
(m.o.i.). At 48 h after infection, cells were serum starved for
16 h (panel A) or cells were treated with indicated concentrations
of rapamycin or vehicle (panel B, DMSO, final 0.1%) for 4 h after
serum starvation for 16 h. Cell lysates were analyzed by
electrophoresis and immunoblotting with anti-IRS-1, antiphospho-Ser473
Akt, antiphospho-Ser411 p70 S6 kinase or antiphospho-Thr202/Tyr204 MAP
kinase antibody. Neither infection of each adenovirus vector nor
treatment with rapamycin had any effect on protein amounts of Akt or
p70 S6 kinase as assessed by non-phospho-specific antibodies (data not
shown). IRS-1 protein levels were analyzed by densitometry and
means ± SE of three independent experiments are shown
(B, lower panel).
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Proteasome Inhibitors Block Insulin-Induced Degradation but Not the
Mobility Shift of IRS-1
To explore the mechanism of insulin-induced degradation of IRS-1,
we next examined the effects of proteasome inhibitors on
insulin-induced mobility shift and degradation of IRS-1 (Fig. 5
). Cells were pretreated with
lactacystin (33) for 60 min, followed by stimulation with insulin for
either 1 or 4 h. Lactacystin almost completely inhibited
insulin-induced degradation of IRS-1 at 4 h (Fig. 5
). At 1 h
after insulin stimulation, the inhibitor did not affect the
insulin-induced mobility shift of IRS-1 (Fig. 5
). Moreover, the
mobility shift did not decrease even at 4 h after insulin
stimulation (Fig. 5
). Less specific inhibitors for the proteasome,
MG132 and proteasome inhibitor I (PSI), had similar effects on
IRS-1 degradation and mobility shift (data not shown).
Insulin-stimulated p70 S6 kinase phosphorylation was not affected by
lactacystin at 1 h and was enhanced at 4 h after stimulation
(Fig. 5
), indicating that the inhibitory effect of lactacystin on
insulin-induced degradation of IRS-1 was not mediated through
inhibition of the rapamycin- sensitive pathway, and that inhibition
of IRS-1 degradation may augment insulin signaling.

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Figure 5. Effects of Lactacystin on Electrophoreitc Mobility
Shift and Degradation of IRS-1
Fully differentiated 3T3-L1 adipocytes were serum starved for 16 h
and pretreated with 10 µM lactacystin or vehicle (DMSO,
final 0.1%) for 60 min before stimulation without or with 20
nM insulin for 1 or 4 h. Cell lysates were analyzed by
electrophoresis and immunoblotting with anti-IRS-1 or
antiphospho-Ser411 p70 S6 kinase antibody. Protein amounts of p70 S6
kinase, as assessed by immunoblotting with non-phospho-specific
antibody were not affected by the treatment (data not shown). A
representative of three independent experiments is shown.
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Rapamycin and Lactacystin Enhance Insulin-Induced Phosphorylation
of Akt
To evaluate the functional consequences of the IRS-1 mobility
shift and IRS-1 degradation, we determined the effects of rapamycin and
lactacystin on downstream events of insulin signaling. First, we tested
the effects of these inhibitors on phosphorylation of Akt at either 1
or 4 h after insulin stimulation. At 1 h, both rapamycin and
lactacystin increased Akt phosphorylation to a small extent (
25%)
but had larger effects at 4 h (Fig. 6
). Since rapamycin inhibited both the
electrophoretic mobility shift and degradation of IRS-1,
whereas lactacystin inhibited degradation alone, these
observations suggest that the effect of rapamycin on Akt
phosphorylation may be through inhibition of IRS-1 degradation, rather
than inhibition of IRS-1 Ser/Thr phosphorylation.

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Figure 6. Effects of Rapamycin or Lactacystin on
Insulin-Stimulated Phosphorylation of Akt
Fully differentiated 3T3-L1 adipocytes were serum starved for 16 h
and pretreated with 4 nM rapamycin for 30 min (panel A), 10
µM lactacystin for 60 min (panel B) or vehicle (DMSO,
final 0.1%) before stimulation without or with 20 nM
insulin for indicated time periods. Cell lysates were analyzed by
electrophoresis and immunoblotting with anti-phospho-Ser473 Akt
antibody. Protein amounts of Akt as assessed by immunoblotting with
non-phospho-specific Akt antibody were not affected by the treatment
(data not shown). Immunoblots of three independent experiments were
quantitated by a densitometer. The values were corrected for those of
control cells stimulated with insulin for 1 h. Means ±
SE are shown as bar graphs.
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Lactacystin Causes Sustained Activation of Insulin Signaling
To assess the role of IRS-1 degradation in insulin action, we
examined the effect of lactacystin on insulin signaling during
prolonged stimulation. Cells were pretreated with lactacystin and
stimulated with insulin for various time periods up to 12 h. IRS-1
protein levels, tyrosine phosphorylation of IRS-1, association of p85
with IRS-1, phosphorylation status and protein levels of Akt, p70 S6
kinase and MAP kinase and protein levels of p85, insulin receptor, and
GLUT4 were determined by immunoblot analysis of the cell lysates or
anti-IRS-1 antibody immunoprecipitates (Fig. 7
). The results show
that progressive loss of IRS-1 protein during prolonged insulin
stimulation was almost completely inhibited by lactacystin (Fig. 7A
).
In lactacystin-treated cells, the mobility shift of IRS-1 did not
decrease during the stimulation period (Fig. 7A
). Lactacystin
significantly inhibited the progressive decrease of pp185 tyrosine
phosphorylation (Fig. 7A
). Similar results were obtained for protein
levels and tyrosine phosphorylation of IRS-1 (Fig. 7B
). The decrease in
the amount of IRS-1-associated p85 was also significantly inhibited by
lactacystin (Fig. 7B
), while total cellular p85 was not affected by
prolonged insulin treatment or by lactacystin (Fig. 7C
). The decrease
in phosphorylation of Akt, p70 S6 kinase, and MAP kinase during
prolonged insulin stimulation, as assessed by immunoblotting with
phosphospecific antibodies, was also significantly inhibited by
lactacystin (Fig. 7A
). The protein amounts of Akt, p70 S6 kinase and
MAP kinase, insulin receptor, and GLUT4 did not change during prolonged
insulin treatment and were not affected by lactacystin (Fig. 7C
).
Therefore, the effect of lactacystin on down-regulation of Akt, p70 S6
kinase, and MAP kinase activation during chronic insulin stimulation
correlated with the effect on IRS-1 degradation.


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Figure 7. Effects of Lactacystin on Insulin Signaling during
Prolonged Stimulation
Fully differentiated 3T3-L1 adipocytes were serum starved for 16 h
and pretreated with 10 µM lactacystin or vehicle (DMSO,
final 0.1%) for 60 min before stimulation without or with 20
nM insulin for the indicated time periods. A, Cell lysates
were analyzed by electrophoresis and immunoblotting with anti-IRS-1,
antiphosphotyrosine, antiphospho-Ser473 Akt, antiphospho-Thr308 Akt,
antiphospho-Ser411 p70 S6 kinase, antiphospho-Thr389 p70 S6 kinase, or
antiphospho-Thr202/Tyr204 MAP kinase antibody. Data were analyzed by
denstitometry and means ± SE of three independent
experiments are shown (lower panels: open symbols,
without lactacystin; closed symbols, with lactacystin).
B, Cell lysates were immunoprecipitated with anti-IRS-1 antibody, and
the immune complexes were then analyzed by electrophoresis and
immunoblotting with anti-IRS-1, antiphosphotyrosine, or anti-p85
antibody. Data were analyzed by denstitometry, and means ±
SE of three independent experiments are shown (lower
panels: open symbols, without lactacystin; closed
symbols, with lactacystin). C, Protein amounts of Akt, p70 S6
kinase, MAP kinase, p85, insulin receptor (IR), and GLUT4 were assessed
by immunoblotting with the indicated non-phospho-specific antibodies. A
representative of three independent experiments is shown.
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Rapamycin but Not Lactacystin Enhances Insulin-Stimulated
2-Deoxyglucose (2-DOG) Uptake
Finally, we examined the effect of rapamycin or lactacystin on
insulin-stimulated 2- DOG uptake. Cells were pretreated with rapamycin
or lactacystin and stimulated with insulin for various time periods,
followed by measurement of 2-DOG uptake. Rapamycin treatment enhanced
2-DOG uptake by 34% and 52% at 1 and 4 h after insulin
stimulation, respectively (Fig. 8
). Thus,
the effect of rapamycin on 2-DOG uptake was similar to the effects of
rapamycin or lactacystin on Akt phosphorylation (Fig. 6
). In contrast,
lactacystin had no significant effect on insulin-stimulated 2-DOG
uptake at any time point examined (Fig. 8
).

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Figure 8. Effects of Rapamycin or Lactacystin on
Insulin-Stimulated 2-DOG Uptake
Fully differentiated 3T3-L1 adipocytes were serum starved for 16 h
and pretreated with vehicle (DMSO, final 0.1%)
(circle), 4 nM rapamycin for 30 min
(triangle) or 10 µM lactacystin for 60 min
(square) before stimulation without or with 20
nM insulin for the indicated time periods.
[3H]2-DOG uptake was then measured as described in
Materials and Methods. Means ± SE of
six independent experiments are shown (*, P <
0.05; **, P < 0.01).
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DISCUSSION
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The current results show that a rapamycin-sensitive pathway,
downstream of PI 3-kinase, mediates the insulin-induced electrophoretic
mobility shift and degradation of IRS-1. This is based on the
observation that two structurally unrelated PI 3-kinase inhibitors, as
well as rapamycin, inhibited these effects of insulin. Moreover, the
finding that activation of the PI 3-kinase pathway by expression of
p110CAAX was sufficient to induce both of these
phenomena in a rapamycin-sensitive manner further supported the role of
PI 3-kinase and places the rapamycin-sensitive pathway downstream of PI
3- kinase. The dependence of the insulin-induced IRS-1 mobility
shift on PI 3-kinase and a rapamycin-sensitive pathway agrees with the
previous finding that PDGF-induced Ser/Thr phosphorylation of IRS-1 was
inhibited by PI 3-kinase inhibitors (24). It is also consistent with
the recent observation that attenuation of insulin-stimulated tyrosine
phosphorylation of IRS-1 by pretreatment with PDGF or activation of Akt
was prevented by rapamycin (25). Therefore, it seems likely that
activation of PI 3- kinase either by insulin or PDGF induces
Ser/Thr phosphorylation of IRS-1 through a common rapamycin-
sensitive pathway, which is possibly mediated by activation of Akt.
Rapamycin acts by binding to mTOR as a gain-of-function complex with
FKBP12, thus compromising the catalytic activity of mTOR (34, 35).
Therefore, mTOR itself, or a Ser/Thr kinase regulated by mTOR, may be
responsible for the insulin-induced Ser/Thr phosphorylation of IRS-1.
Alternatively, it is possible that a Ser/Thr protein phosphatase that
is negatively regulated by mTOR (36) may play a role in
dephosphorylation of IRS-1.
Evidence has indicated that Ser/Thr phosphorylation of IRS-1 induced by
various agents or mediators inhibits insulin-stimulated tyrosine
phosphorylation of IRS-1, leading to attenuation of insulin signaling
(12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25). Our data, however, suggest that Ser/Thr phosphorylation of
IRS-1 caused by insulin, which is responsible for the insulin-mediated
mobility shift, may not significantly affect IRS-1 tyrosine
phosphorylation or downstream insulin signaling, since tyrosine
phosphorylation of IRS-1 did not seem to be significantly affected by
inhibition with PI 3-kinase inhibitors or rapamycin, and the effects of
rapamycin and lactacystin on Akt phosphorylation were not significantly
different. On the other hand, the insulin-induced mobility shift occurs
slowly as compared with tyrosine phosphorylation of IRS-1 (Fig. 1
).
Therefore, when IRS-1 is already phosphorylated on tyrosine residues,
Ser/Thr phosphorylation induced by insulin may not significantly affect
tyrosine phosphorylation of IRS-1 or downstream insulin signaling.
Thus, the current data are still consistent with the idea that Ser/Thr
phosphorylation of IRS-1 induced through a rapamycin-sensitive pathway
by other agents or mediators, such as PDGF, Akt, or PI 3-kinase
(23, 24, 25, 32), may impair insulin-stimulated tyrosine phosphorylation of
IRS-1. It is also possible that increases in tyrosine phosphorylation
of IRS-1 and downstream insulin effects in response to restimulation
with insulin may be attenuated when Ser/Thr phosphorylation of IRS-1 is
already induced by insulin.
We have shown that a highly specific inhibitor for the proteasome,
lactacystin, as well as less specific inhibitors, almost completely
prevented insulin-induced degradation of IRS-1. This result is
consistent with the recent report by Sun et al. (37).
Furthermore, our finding that insulin-induced degradation of IRS-1 was
also dependent on PI 3-kinase and the rapamycin-sensitive pathway
strongly suggests that insulin-stimulated Ser/Thr phosphorylation of
IRS-1, which seems to be mediated by mTOR, may be required for its
subsequent degradation by the proteasome. Consistent with this notion,
the data showed that the mobility shift of IRS-1 did not diminish
during prolonged insulin treatment in the presence of proteasome
inhibitors, suggesting that the proteasome may specifically degrade
Ser/Thr-phosphorylated IRS-1. The vast majority of known protein
substrates of the proteasome must be modified by the covalent
attachment of a polyubiquitin chain, which serves as a
substrate-targeting and recognition signal for the proteasome, and, in
many instances, signal-induced phosphorylation is a prerequisite for
subsequent ubiquitination of the substrate protein (38, 39, 40, 41). Therefore,
phosphorylation of IRS-1 mediated by the rapamycin-sensitive pathway
may lead to ubiquitination of IRS-1, which is required for degradation
by the proteasome. Alternatively, Ser/Thr phosphorylation of IRS-1 may
lead to modification of IRS-1 other than ubiquitination or promote
interaction of the already ubiquitinated or modified IRS-1 with a
component of the proteasomal degradation machinery. The recent report
that IRS-1 was polyubiquitinated, even in the basal state, and that
insulin stimulation was without effect on the extent of the
ubiquitination (37) agrees with the latter possibilities.
Our data demonstrate that degradation of IRS-1 by the proteasome plays
a major role in down-regulation of insulin signaling during prolonged
stimulation. Thus, blockade of IRS-1 degradation by either rapamycin or
lactacystin resulted in increases in tyrosine phosphorylation of IRS-1
and significant enhancement of Akt phosphorylation at 4 h after
insulin stimulation. Furthermore, blockade of IRS-1 degradation by
lactacystin significantly inhibited the progressive decrease of
IRS-1-associated p85 and progressive inactivation of Akt, p70 S6
kinase, and MAP kinase until 12 h after insulin stimulation. The
finding that chronic insulin treatment or lactacystin were without
effect on protein levels of Akt, p70 S6 kinase, and MAP kinase and
other components of insulin signaling cascade, including insulin
receptor, p85, and GLUT4, further supports the contribution of IRS-1
degradation to down-regulation of insulin action during prolonged
stimulation.
Although blockade of IRS-1 degradation by lactacystin enhanced insulin
effects such as activation of Akt, p70 S6 kinase, and MAP kinase,
lactacystin was without effect on 2-DOG uptake, whereas this was
significantly enhanced by rapamycin. The reason for the differential
effects of lactacystin and rapamycin on 2-DOG uptake is not clear. One
explanation is that IRS-1 may not play a key role in insulin-stimulated
glucose transport, consistent with several previous reports (42, 43, 44, 45).
In this scenario, rapamycin may enhance insulin-stimulated 2-DOG uptake
by a mechanism independent of IRS-1. Alternatively, the observation
that the mobility shift of IRS-1 did not decline during prolonged
insulin stimulation in the presence of lactacystin raises the
possibility that inhibition of proteasomal degradation results in the
accumulation of modified forms of IRS-1. These forms of IRS-1 may not
target to the intracellular location appropriate for eliciting GLUT4
translocation, but still can couple to other effects of insulin
including activation of Akt, p70 S6 kinase, and MAP kinase.
mTOR controls protein synthesis by regulating the translational
components, 4E-BP1 and p70 S6 kinase (9). Recent evidence suggests that
mTOR functions as a sensor of amino acids and balances nutrient supply
and protein synthesis (35, 46, 47). Since one of the major metabolic
actions of insulin is to stimulate uptake of amino acids, it is
tempting to speculate that modulation of insulin action by the
rapamycin-sensitive pathway may be a part of an adaptational response
to nutrient availability.
In summary, these results indicate that both insulin-stimulated Ser/Thr
phosphorylation of IRS-1, which is seen as an electrophoretic mobility
shift, and insulin-induced degradation of IRS-1 are mediated by a
rapamycin-sensitive pathway that is downstream of PI 3-kinase and
independent of the ras/MAP kinase pathway. The results further show
that the pathway leads to degradation of IRS-1 by the proteasome, which
plays a major role in down-regulation of insulin action during
prolonged insulin stimulation.
 |
MATERIALS AND METHODS
|
---|
Materials
Anti-IRS-1 antibody and antirat p85 subunit of PI 3-kinase
antibody were purchased from Upstate Biotechnology, Inc.
(Lake Placid, NY). Horseradish peroxidase (HRP)-conjugated monoclonal
antiphosphotyrosine antibody (PY20H) was purchased from
Transduction Laboratories, Inc. (Lexington, KY). LY294002,
wortmannin, and rapamycin were obtained from Sigma (St.
Louis, MO). PD98059 and phospho-specific and non-phospho-specific
antibodies against Akt, p70 S6 kinase, and MAP kinase were obtained
from New England Biolabs, Inc. (Beverly, MA). Monoclonal
anti-GLUT4 antibody (1F8) was from East Acres Biologicals
(Southbridge, MA). Antiinsulin receptor ß-subunit antibody and
HRP-conjugated antimouse and rabbit IgG antibodies were purchased from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Porcine
insulin was kindly provided by the Lilly Research Laboratories
(Indianapolis, IN). Lactacystin, MG132, and PSI were obtained from
Calbiochem (La Jolla, CA). Polyvinylidene difluoride
(PVDF) membrane was obtained from Millipore Corp.
(Bedford, MA). DMEM and FCS were purchased from Life Technologies, Inc. (Gaithersburg, MD). Protein G-Sepharose was
purchased from Pharmacia Biotech (Uppsala, Sweden).
Electrophoresis reagents were from Bio-Rad Laboratories, Inc. (Hercules, CA). [1, 2, -3H]
2-Deoxyglucose was from NEN Life Science Products
(Boston, MA). All other reagents and chemicals were from standard
suppliers.
Cell Culture
3T3-L1 fibroblasts obtained from American Type Culture Collection (ATCC, Manassas, VA) were cultured in
DMEM high glucose containing 100 U/ml penicillin, 100 mg/ml
streptomycin, and 10% FCS in 10% CO2 atmosphere
and induced to differentiate into adipocytes as described previously
(48). 3T3-L1 adipocytes were used between 14 and 19 days after
initiation of differentiation, when more than 95% of the cells
exhibited an adipocyte-like phenotype.
Human embryonic kidney 293 cells obtained from ATCC were
cultured in DMEM high glucose containing 100 U/ml penicillin, 100 mg/ml
streptomycin, and 10% FCS in 5% CO2
atmosphere.
Infection of Recombinant Adenovirus Vectors
The recombinant adenoviruses, Ad5-p110CAAX
containing bovine p110
cDNA with the CAAX motif at the COOH terminus
and Ad5-CT that has no insert, were described previously (32). They
were amplified in 293 cells and viral stock solutions with viral
titer > 108 plaque forming units/ml were
prepared. 3T3-L1 adipocytes were infected with the vectors by
incubating the cells at indicated multiplicity of infection (m.o.i.) of
viral stock solution in DMEM containing 2% heat-inactivated FCS
overnight. The medium was replaced with DMEM containing 10% FCS, and
the cells were used 48 h after infection.
Immunoprecipitation and Immunoblotting
Cells were solubilized at 4 C in an ice-cold buffer containing
20 mM Tris, pH 7.5, 140 mM NaCl, 1%
Nonidet-P40, 1 mM EDTA, 2.5 mM sodium
pyrophosphate, 1 mM ß-glycerophosphate, 2 mM
Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 50 U/ml aprotinin, 1 µM
leupeptin and 50 mM NaF, and centrifuged at 15,000 x
g for 30 min to remove insoluble material. For
immunoprecipitation of IRS-1, the cell lysates (500 µg protein) were
incubated with anti-IRS-1 antibody (2 µg) for 16 h at 4 C,
followed by incubation with protein G-Sepharose for 2 h. The cell
lysates or immunoprecipitates were boiled in Laemmli buffer, resolved
by SDS-PAGE with 7.5% or 12% acrylamide gels, and transferred to PVDF
membranes. When antibody to phosphotyrosine was used, the membrane was
blocked in TBS-T (10 mM Tris, pH 7.6, 150
mM NaCl, 0.1% Tween 20) containing 4% BSA
overnight at 4 C and incubated with HRP-conjugated antiphosphotyrosine
antibody (PY20H) for 1 h at room temperature. For immunoblotting
with anti-IRS-1, p85, insulin receptor, or GLUT4 antibody, the membrane
was blocked with TBS-T containing 5% nonfat dry milk and incubated
with the indicated antibody for 1 h at room temperature, followed
by incubation with HRP-conjugated secondary antibody. For detection of
phospho- or non-phospho-Akt, p70 S6 kinase, or MAP kinase, the membrane
was blocked with TBS-T containing 5% nonfat dry milk and incubated
with the indicated antibody for 16 h at 4 C, followed by
incubation with HRP-conjugated secondary antibody. The proteins were
visualized by enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech).
2-DOG Uptake
Before the assay of [3H]2-DOG uptake,
the cells were deprived of serum for 16 h and were stimulated with
20 nM insulin for the indicated period of time. Unlabeled
2-DOG and [3H]-2-DOG (0.1 mM , 0.74
kBq/well) were added to the cells in the KRP-HEPES buffer (10
mM HEPES, pH 7.4, 131.2 mM NaCl, 4.7
mM KCl, 1.2 mM MgSO4, 2.5
mM CaCl2, 2.5 mM
NaH2PO4) with 1% BSA at 37
C and incubated for 4 min. Reaction was stopped by adding 10
µM cytochalasin B and washing cells with ice-cold PBS
three times. The cells were solubilized in 1 ml of 0.2% SDS and 0.2
N NaOH. The radioactivity was quantitated in a liquid
scintillation counter. The values of nonspecific uptake and absorption
were determined by [3H]2-DOG uptake in the
presence of 10 µM cytochalasin B, and the results were
corrected for these values. Nonspecific uptake and absorption were
always less than 10% of total uptake.
Statistical Analysis
Data were analyzed by Students t test. P
values < 0.05 were considered significant.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Tetsuro Haruta, MD, Ph.D., First Department of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama, 930-0194, Japan.
This study was supported in part by a grant-in-aid for Scientific
Research from the Ministry of Education, Science, Sports and Culture,
Japan (to T.H.) and by NIH Grant DK-33651 to (J.M.O.).
Received for publication October 12, 1999.
Revision received February 7, 2000.
Accepted for publication February 10, 2000.
 |
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