From INSERM U145, IFR50, Faculté de Médecine, 06107 Nice Cedex 2, France
Received for publication, September 3, 2002, and in revised form, February 11, 2003
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ABSTRACT |
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Impaired glucose tolerance precedes type 2 diabetes and is characterized by hyperinsulinemia, which develops to
balance peripheral insulin resistance. To gain insight into the
deleterious effects of hyperinsulinemia on skeletal muscle, we studied
the consequences of prolonged insulin treatment of L6 myoblasts on
insulin-dependent signaling pathways. A 24-h long insulin
treatment desensitized the phosphoinositide 3-kinase
(PI3K)/protein kinase B (PKB) and p42/p44 MAPK pathways toward a second
stimulation with insulin or insulin-like growth factor-1 and led to
decreased insulin-induced glucose uptake. Desensitization was
correlated to a reduction in insulin receptor substrate (IRS)-1 and
IRS-2 protein levels, which was reversed by the PI3K inhibitor
LY294002. Co-treatment of cells with insulin and LY294002, while
reducing total IRS-1 phosphorylation, increased its phosphotyrosine
content, enhancing IRS-1/PI3K association. PDK1, mTOR, and MAPK
inhibitors did not block insulin-induced reduction of IRS-1, suggesting
that the PI3K serine-kinase activity causes IRS-1 serine
phosphorylation and its commitment to proteasomal degradation.
Contrarily, insulin-induced IRS-2 down-regulation occurred via a
PI3K/mTOR pathway. Suppression of IRS-1/2 down-regulation by LY294002
rescued the responsiveness of PKB and MAPK toward acute insulin
stimulation. Conversely, adenoviral-driven expression of constitutively
active PI3K induced an insulin-independent reduction in IRS-1/2 protein
levels. IRS-2 appears to be the chief molecule responsible for MAPK and
PKB activation by insulin, as knockdown of IRS-2 (but not IRS-1) by RNA
interference severely impaired activation of both kinases. In summary,
(i) PI3K mediates insulin-induced reduction of IRS-1 by phosphorylating
it while a PI3K/mTOR pathway controls insulin-induced reduction of
IRS-2, (ii) in L6 cells, IRS-2 is the major adapter molecule linking
the insulin receptor to activation of PKB and MAPK, (iii) the mechanism
of IRS-1/2 down-regulation is different in L6 cells compared with
3T3-L1 adipocytes. In conclusion, the reduction in IRS proteins via
different PI3K-mediated mechanisms contributes to the development of an
insulin-resistant state in L6 myoblasts.
Type 2 diabetes is caused by a progressive decrease in insulin
action and gradual development of chronic hyperglycemia. Initial peripheral insulin resistance, i.e. the failure of adipose
and muscle tissues to properly dispose of circulating glucose, and the
failure of liver to control glucose production, is compensated by
increased insulin secretion from pancreatic Activation of the insulin receptor
(IR)1 by hormone binding
activates a number of cellular responses such as translocation of the
glucose transporter GLUT4 to the plasma membrane in muscle and
adipocytes (3), hepatic glycogen synthesis (4), lipogenesis (5), and
modulation of gene expression (6).
Insulin signaling is initiated by the recruitment of intracellular
molecules to the activated receptor and their ensuing tyrosine phosphorylation. These molecules include IRS-1/2/3/4, Cbl, and the
adapter proteins Shc (7). Phosphorylated IRSs then engage the
phosphoinositide 3-kinase (PI3K) p85/p110 heterodimer by binding the
SH2 domains of the p85 adapter to specific pYMXM
motifs (8). Once captured by IRS, PI3K becomes activated and produces
the lipid second messenger phosphatidylinositol
3,4,5-trisphosphate, which stimulates the serine/threonine
kinase cascade PDK-1-PKB/Akt-p70 S6 kinase (9). PKB/Akt and p70
S6 kinase are downstream kinases central to insulin action (10), the
former controlling GLUT4 translocation, glycogen synthesis, and protein
synthesis (11), and the latter being a key regulator of the growth
promoting action of insulin (12). Other SH2 domain containing proteins,
including Grb2, SHP-2, and Nck associate with IRS to mediate insulin
responses. In particular, Grb2 links insulin receptor activation to the
p42/p44 mitogen-activated protein kinase (MAPK) cascade (7).
The occurrence of insulin-induced peripheral insulin resistance has
been attributed to impairment of the tyrosine kinase activity of the
insulin receptor, based on biochemical studies performed on skeletal
muscle biopsies from type 2 diabetic patients (13-15). However, these
findings have been disputed by reports showing normal insulin receptor
function, despite downstream metabolic abnormalities (16-18).
Recently, the elucidation of the signaling pathways
emanating from the activated insulin receptor (10, 19) has paved the
way for several studies demonstrating that intracellular signaling
molecules also substantially contribute to the insulin-resistant phenotype.
IRS-1 protein levels have been shown to be decreased in 3T3-L1
adipocytes chronically exposed to insulin (20). IRS-1 degradation is
triggered by serine/threonine phosphorylation, and is blocked by the
PI3K inhibitor LY294002 (21, 22). PI3K may control IRS-1
serine/threonine phosphorylation either via the action of downstream
serine/threonine kinases, such as mTOR, GSK-3, and atypical protein
kinase Cs, which ultimately phosphorylate IRS-1 (23, 24), or by
directly phosphorylating IRS-1 via its intrinsic protein kinase
activity (25, 26). Once serine/threonine phosphorylated, IRS-1
degradation occurs via the proteasome degradation pathway (27). In
keeping with these studies in cultured cells, is the finding that IRS-1
protein expression is reduced in adipocytes from patients with type 2 diabetes (28) as well as in subjects suffering from insulin resistance
(29). Recently, proteasome-mediated IRS-2 degradation has also been
demonstrated in insulin/IGF-1-treated 3T3-L1 adipocytes, Fao hepatoma
cells, and mouse embryo fibroblasts (30).
Downstream of IRS proteins, other insulin signaling molecules have been
reported to be deregulated in insulin-induced insulin resistance or
type 2 diabetes. PKB activation is decreased in skeletal muscle and
adipose tissue from db/db mice as compared with non-diabetic controls
(31). GLUT4 expression is lower in insulin-resistant individuals (32),
and in L6 myotubes overexpressing the transporter GLUT4, the
insulin-induced GLUT4 translocation to the plasma membrane is reduced
after sustained exposure to high glucose and insulin (33). Thus,
deterioration of the insulin receptor-signaling pathway at different
levels accounts for the evolution from an insulin-resistant state to
type 2 diabetes.
Here we evaluate the changes in insulin signaling pathways after a
prolonged exposure of L6 muscle cells to insulin and we investigate the
underlying molecular mechanisms. We demonstrate that, upon prolonged
insulin treatment (mimicking hyperinsulinemia), both the PI3K/PKB and
the MAPK signaling cascades are down-regulated and cellular levels of
the two major insulin receptor docking proteins, IRS-1 and IRS-2, are
decreased. Finally, we show that PI3K/PKB and MAPK down-regulation is
causally related to the decrease in IRS-1/2, that PI3K is a key
upstream molecule controlling IRS-1/2 degradation via different
mechanisms, and that IRS-2 appears to be the functionally most relevant
adapter in insulin signal transmission in L6 cells.
Materials--
Cell culture solutions and supplements, reagents
for SDS-PAGE, and Protein A-Sepharose were from Invitrogen
(Carlsbad, CA). Polyvinylidene difluoride membranes for immunoblotting
were from Millipore (Bedford, MA). ECL reagents,
[ Cell Culture--
Rat L6 myoblasts were obtained from the
American Type Culture Collection (ATCC, Rockville, MD). L6 were grown
in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
(v/v) fetal calf serum (FCS), penicillin, 100 units/ml, and
streptomycin, 100 µg/ml (Invitrogen). Cells were seeded in 6-well
plates for Western blotting experiments or in 10-cm diameter plates for
immunoprecipitation experiments. 24-h insulin treatments were started
at 70-80% confluence in serum-free medium.
Immunoblotting, Immunoprecipitations, PI3K Assays, and
2-Deoxyglucose Uptake--
After treatment with insulin or inhibitors,
cells were lysed in buffer A (20 mM Tris-HCl, 138 mM NaCl, 2.7 mM KCl, 5% (v/v) glycerol, 1 mM sodium-o-vanadate, 20 µM
leupeptin, 18 µM pepstatin, 1% (v/v) Nonidet P-40, 5 mM EDTA, 20 mM NaF, pH 8.0; 0.4 ml/plate for
6-well plates and 1 ml/plate for 10-cm diameter plates). Lysates were
kept on ice for 15 min and the insoluble material was removed by
centrifugation at 13,000 × g for 15 min. Protein
concentration was determined by the Bio-Rad colorimetric assay
(Bio-Rad).
Cell lysates (10 µg of protein) were separated by SDS-PAGE,
transferred to Immobilon-P polyvinylidene difluoride membranes, and
blocked in 5% (w/v) bovine serum albumin. Membranes were probed with
anti-phospho-Ser-473 PKB (New England Biolabs, Beverly, MA), anti-PKB
(provided by B. Hemmings, FMI, Basel, Switzerland), anti-phospho-MAPK p42/p44, anti-MAPK p42/p44 (Promega, Madison, WI), anti-IRS-1, anti-IRS-2, 4G10 anti-phosphotyrosine (Upstate Biotechnology, Lake
Placid, NY), and anti-IR
For immunoprecipitation, lysates were incubated at 4 °C for 30 min
with polyclonal antibodies to IRS-1 or to IR Orthophosphate Metabolic Labeling--
When at 70-80%
confluence, L6 myoblasts (one 10-cm diameter plate/condition) were
incubated for 24 h in 5 ml of phosphate-free DMEM in the presence
of 1 µM insulin and inhibitors as described in figure
legends. Cells were labeled for the last 3 h with 0.5 mCi/ml
[32P]orthophosphate and IRS-1-associated radioactivity
was evaluated in IRS-1 immunoprecipitates by SDS-PAGE separation
followed by phosphorimaging of the dried gel.
Generation of Recombinant Adenoviruses--
Adenoviruses
expressing p110 Northern Blotting and RNA Interference--
Total RNA was
extracted from cells using Trizol reagent (Invitrogen). 15 µg of
RNA were resolved by electrophoresis on a 1.2% agarose-formaldehyde
gel. RNA was transferred to a BrightStar-Plus positively charged nylon
membrane (Ambion, Austin, TX). Probes were labeled with
[
For RNA interference experiments, L6 were grown in DMEM + 10% (v/v)
FCS in 24-wells plates. At >60% confluence medium was replaced with
300 µl of DMEM + 5% (v/v) FCS and cells were transfected by the
calcium phosphate method with double stranded RNA oligonucleotides at a
concentration of 50 nM (Proligo, Dusseldorf, Germany).
Briefly, 0.3 µl of 50 µM dsRNA oligonucleotides were
mixed with 7.5 µl of water, 7.5 µl of calcium solution (0.5 M CaCl2, 50 mM HEPES, pH 7.0), and
15 µl of phosphate solution (0.075 mM
Na2HPO4, 0.075 mM
NaH2PO4, 0.28 M NaCl, 0.05 M HEPES, pH 7.0). The mixture was left for 10 min at room
temperature to form a precipitate and subsequently added to the cells.
Medium was replaced 12-16 h later with DMEM + 5% (v/v) FCS. Prior to
insulin treatment, cells were starved overnight in DMEM. In L6, RNA
interference (RNAi) occurred after calcium phosphate transfection, but
not after transfection with FuGENE or simple addition of short
interfering double stranded RNA (siRNA) to the culture medium. DsRNA
oligonucleotides used in this study encompassed nucleotides 895-913
and 1180-1198 of the published IRS-1 (X58375) and IRS-2 (XM146235)
sequences. Two deoxythymine residues were added 3' to each strand.
DsRNAs annealing was done in annealing buffer (10 mM
Tris-HCl, 50 mM NaCl, 1 mM EDTA, pH 8) by
heating the complementary single-stranded RNAs at 90 °C for 2 min
followed by slow cooling to <30 °C.
Prolonged Insulin Treatment Desensitizes the PI3K/PKB
and MAPK Signaling and Decreases Insulin-induced 2-Deoxyglucose
Uptake--
To investigate the alterations in intracellular signaling
caused by a prolonged insulin treatment in muscle cells, L6 myoblasts were exposed for 24 h to increasing insulin concentrations,
followed or not by a second treatment with 1 µM hormone
for 10 min.
To evaluate the activation of the PI3K and the MAPK signaling,
IRS-1-associated PI3K activity was assayed in IRS-1 immunoprecipitates, and MAPK activation was evaluated by immunoblotting with antibodies to
phospho-MAPK. Responsiveness of both PI3K and MAPK to a 10-min exposure
to 1 µM insulin was diminished to a similar extent, and in a dose-dependent manner, in cells subjected to a 24-h
insulin pre-treatment (Fig. 1,
A and B, empty bars), indicating
desensitization of the insulin signaling pathways. On the contrary,
whereas an IRS-1-associated PI3K activity could be detected after the
24-h insulin treatment, no residual MAPK activity was present (Fig. 1,
A and B, full bars). Thus, insulin
chronically activates PI3K at a submaximal level, although MAPK
activation is transient and becomes undetectable after 4 h insulin
stimulation (data not shown).
To determine whether IRS-1-associated PI3K activity relays a downstream
signal in intact cells, we next evaluated the activation of PKB by
immunoblotting with antibodies to the active form of PKB (Ser-473 PKB).
PKB activation paralleled IRS-1-associated PI3K activity under all the
conditions tested (Fig. 2, A
and B). Likewise, p70 S6 kinase activation showed an
activation pattern similar to that of PKB, as judged by insulin-induced
band shift (data not shown). A time course evaluation of the decline in
insulin responsiveness of PKB and MAPK demonstrated that the
down-regulation of both signaling pathways occurred in a
time-dependent fashion, with the MAPK signaling being
down-regulated more rapidly than PKB (Fig. 2C).
The correlation between the IRS-1-associated PI3K activity and the PKB,
p70 S6 kinase activation levels observed indicates that the
IRS-1-associated PI3K activity mirrors the PI3K activity in
vivo. Furthermore, a 24-h insulin treatment, although provoking a
Down-regulation of insulin signaling has been proposed to result from
alterations in both insulin receptor and postreceptor signaling (38).
To evaluate whether the down-regulation observed in PI3K/PKB and MAPK
signaling was because of insulin-induced IR degradation we quantified
the total amount of IR in starved or 24-h insulin-treated L6 myoblasts
by immunoblotting with antibodies to IR. As already reported in
myotubes (33), the IR levels and insulin-induced IR tyrosine
phosphorylation decreased by ~50% as compared with untreated cells
following a 24-h exposure to 1 µM insulin (Fig.
3). Given that low receptor occupancy can
be sufficient to relay a biological effect (39), deregulation of intracellular component(s) of the insulin signaling cascade might also
contribute to the observed down-regulation.
Insulin/IGF-1-treated L6 Cells Retain Responsiveness to
Activation by FCS, and PDGF
Whereas cross-reactivity occurs between insulin and IGF-1 toward their
corresponding receptors, FCS and PDGF are not known to interfere with
IR or IGF-1R. Consistent with this, MAPK activation by FCS was
sustained after a 24-h insulin pretreatment (Fig. 4C), indicating that, after prolonged insulin/IGF-1 exposure, the PI3K/PDK-1 and Raf-MEK signaling modules remain responsive to treatment by agonists other than insulin/IGF-1. Because PKB and MAPK are targets of
virtually all activated receptor-tyrosine kinases, we evaluated whether
insulin signaling down-regulation is specifically achieved by
insulin/IGF-1 pre-exposure or if it could also be caused by prolonged
exposure to an unrelated receptor-tyrosine kinase agonist. When we
exposed cells for 24 h to PDGF and subsequently treated acutely
with either insulin or PDGF, we observed homologous desensitization of
PKB and MAPK (complete and Desensitization of Insulin Signaling Is Associated with a
PI3K-dependent Decrease in IRS-1 and IRS-2 Protein
Levels--
Previous studies on Zucker fatty rats and ob/ob mice, an
hyperinsulinemic and insulin-resistant rodent models, respectively, show decreased IRS-1 expression levels in skeletal muscle (41, 42).
Likewise, IRS-1 was found to be down-regulated in adipose tissue from
diabetic patients (28) and in 3T3-L1 adipocytes treated chronically
with insulin (43).
Therefore, we evaluated the protein levels of IRS-1 and IRS-2 in 24-h
insulin-treated L6 myoblasts. Both IRS-1 and IRS-2 decreased after a
24-h exposure to insulin in a dose-dependent manner,
reaching 30% of untreated controls with 1 µM insulin
(Fig. 5, A and B). The decrease in IRS-1 was post-transcriptional, because we did not
observe decreased expression of the IRS-1 mRNA after insulin treatment, although GLUT-1 mRNA was induced, confirming the
insulin-induced transcriptional effects (Fig. 5C (44)). We
also evaluated the protein levels of p42/p44 MAPK and PKB. p42/p44
expression was unaltered by prolonged insulin treatment (Fig.
5A). On the contrary, PKB expression increased up to
2.4-fold in a concentration-dependent manner (Fig. 5,
A and B).
Insulin-induced degradation of IRS-1 is mediated by the proteasome
degradation pathway (27, 45) following serine/threonine phosphorylation
(46). Using pharmacological inhibitors, we attempted to determine which
kinase(s) might lead to IRS-1 serine/threonine phosphorylation.
Co-treatment of cells with insulin and LY294002 resulted in the
inhibition of IRS-1 degradation and in a lowering of its apparent
molecular mass, indicating a decreased serine/threonine phosphorylation. Likewise, LY294002 prevented insulin-induced IRS-2
degradation (Fig. 6A). The
inhibitory action of LY294002 throughout the 24-h treatment was
confirmed by immunoblot analysis with antibodies against active PKB
(Fig. 6B). That LY294002 blocks insulin-induced IRS-1
down-regulation indicates that PI3K regulates IRS-1 serine/threonine
phosphorylation, either directly, or via activation of
downstream kinase(s) and/or inhibition of phosphatases. To assess the
role of PI3K-activated kinases in regulating IRS-1 down-regulation,
24-h insulin-stimulated L6 cells were simultaneously treated with
the anti-proliferative agent TPCK, which inhibits both PKB
and p70 S6 kinase by disrupting PDK1 signaling (47). In contrast to
LY294002, TPCK, although inhibiting both PKB and p70 S6 kinase
activation (Fig. 6C), was ineffective in blocking insulin-induced IRS-1 down-regulation (Fig. 6C).
To evaluate the potential role of p42/p44 MAPK and mTOR, which have
been reported to phosphorylate IRS-1 (24, 48), we incubated L6
myoblasts with or without 1 µM insulin for 24 h in the presence of either 50 µM LY294002, 50 nM
rapamycin or 10 µM PD98059. In the absence of insulin,
LY294002, but not rapamycin or PD98059, induced a mobility shift toward
a lower apparent molecular mass, indicating that basal IRS-1
phosphorylation is chiefly controlled by PI3K. Moreover, LY294002, but
not rapamycin or PD98059, blocked IRS-1 degradation (Fig.
6D). On the contrary, treatment of cells with either
LY294002 or rapamycin, irrespective of insulin co-treatment, led to an
induction of IRS-2, indicating that a basal activity of the PI3K-mTOR
pathway suffices to trigger a constitutive IRS-2 degradation pathway.
This observation is in agreement with the work of Simpson et
al. (49) who, by overexpressing the phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase PTEN in PTEN-null breast cancer cells, observed a feedback up-regulation of IRS-2 mRNA and protein level.
To gain further insight into the kinase(s) involved in insulin-induced
IRS-1 degradation, 24-h insulin-treated L6 cells were labeled with
[32P]orthophosphate in the presence of the above
inhibitors and IRS-1 phosphorylation was quantitated in IRS-1
immunoprecipitates. LY294002 and rapamycin, but not PD98059, induced a
decrease in IRS-1 phosphorylation (Fig.
7A, left). Because
LY294002 (but not rapamycin) also inhibited the insulin-induced IRS-1
decrease (Fig. 6, A and D) it is PI3K inhibition that yields the maximal inhibitory effect on IRS-1 phosphorylation (Fig. 7A). Moreover, in 24-h insulin-treated
L6 cells, LY294002 addition (but not addition of other protein kinase inhibitors nor of MG132, a proteasome inhibitor, see legend to Fig. 7)
resulted in increased IRS-1 tyrosine phosphorylation and increased
IRS-1-associated p85
To confirm the role of PI3K in controlling IRS-1/2 down-regulation, L6
myoblasts were infected at increasing multiplicity of infection with
adenovirus expressing a constitutively active PI3K catalytic subunit
(p110 Insulin-induced Desensitization of the PI3K/PKB and MAPK
Pathways Is Reversed by a Blockade of IRS-1/2
Down-regulation--
To demonstrate a direct link between
desensitization of insulin signaling upon prolonged exposure to insulin
and decreased levels of IRS molecules, L6 myoblasts were exposed for
24 h to insulin in the presence of 50 µM LY294002,
thus inhibiting down-regulation of IRS1/2. After 24 h, the
inhibitor was removed and the cells were stimulated with 1 µM insulin, as indicated in Fig.
9. A 24-h pretreatment with
103 nM insulin down-regulated PKB and MAPK
(Fig. 9, lanes 1-4; see also Figs. 1 and 2). However, PKB
could be maximally activated by a 10-min exposure to 1 µM
insulin after a 24-h incubation with 102 or 103
nM hormone, provided that LY294002 was added (Fig. 9).
Likewise, a transient maximal activation of MAPK could be induced after 24 h treatment with 102 nM insulin and
LY294002, and a lower activation could still be observed when
103 nM insulin was administered. The
concomitant occurrence of (i) inhibition of IRS-1/2 down-regulation by
PI3K inhibition, and (ii) activation of PKB and MAPK by acute insulin
stimulation after prolonged insulin/LY294002 treatment, indicates that
the desensitization of insulin signaling depends on the decreased
protein levels of IRS-1/2 proteins.
RNAi-mediated Knockdown Establishes That IRS-2 Is the Major Adapter
Accounting for Insulin-induced Desensitization of the
PI3K/PKB and MAPK in L6 Cells--
To elucidate the
relative contribution that the decrease in IRS-1 versus
IRS-2 plays in the down-regulation of insulin signaling, we took
advantage of the recently described RNAi approach, whereby a protein
can be selectively knocked down by transfection of siRNA (51). Double
stranded siRNAs designed to selectively knockdown IRS-1 or IRS-2 in L6
cells reduced their expression level by >80% (Fig.
10B). Insulin stimulation of
cells knocked down for IRS-1 led to an activation of PKB and MAPK
comparable with that of cells not exposed to siRNA. On the contrary,
elimination of IRS-2 rendered both PKB and MAPK almost unresponsive to
insulin, indicating that the major adapter molecule linking the
activated insulin receptor to downstream PKB and MAPK activation is
IRS-2 in L6 cells (Fig. 10A). Because by knockdown of IRS-2,
we observed a decrease in IRS-1 levels (Fig. 10B) it could
not be ruled out that the impaired activation of PKB and MAPK was
because of the concomitant decrease in IRS-1. To test whether
physiological levels of IRS-1 may overcome the lack of IRS-2, L6 cells
in which RNAi to IRS-2 had been performed were infected at a 10-150
m.o.i. of an adenovirus expressing IRS-1. Under these experimental
conditions, IRS-1 expression was restored, with overexpression at the
highest multiplicity of infection. However, upon insulin stimulation no
rescue of either PKB or MAPK activities was observed (Fig.
10C), indicating that even a fully functional IRS-1 cannot
overcome, for these biological responses, the absence of IRS-2 in L6
cells.
Insulin resistance results from the inability of
insulin-responsive tissues to respond properly to the hormone. This
state is associated with hyperinsulinemia, and when increased insulin secretion becomes insufficient to maintain glucose homeostasis, chronic
hyperglycemia and type 2 diabetes occur.
To investigate the molecular alterations induced by hyperinsulinemia in
muscle cells, we treated L6 cells for 24 h with different hormone
concentrations followed by a second 10-min acute hormone treatment.
These conditions mimic a hyperinsulinemic state, whereby a temporarily
increased insulin secretion is superimposed upon an already elevated
insulin level.
In this system, PI3K recruitment to IRS-1 and activation of PI3K/PKB
and MAPK pathways by acute insulin stimulation were reduced in a
dose-dependent manner by the preceding 24-h insulin
treatment, with a 50% down-regulation of both pathways at
102 nM hormone pretreatment. The responses of
the two pathways to the sole 24-h insulin exposure differed, MAPK
activity being absent and the PI3K/PKB activation reaching a submaximal
level (30-50% of the maximal insulin-stimulated activity) at all
hormone concentrations tested. The 24-h insulin-induced down-regulation
of PI3K/PKB resulted in a decreased acute insulin-induced
[3H]2-deoxyglucose uptake. This reduction is likely to be
because of a defective insulin-induced GLUT4 translocation at the
plasma membrane (33).
Given the debate as to whether down-regulation of insulin receptor
number and/or reduced kinase activity is responsible for determining
the insulin-resistant state (see Introduction), we evaluated the amount
and extent of tyrosine phosphorylation of the insulin receptor (Fig.
3). Blotting of immunoprecipitated insulin receptors with antibodies to
the receptor demonstrated a reduction of ~50% in both receptor
content and receptor tyrosine phosphorylation after a 24-h exposure to
insulin. Given that low receptor occupancy and activation can relay a
biological signal, we investigated whether intracellular downstream
targets might also be involved in the down-regulation of
insulin-induced intracellular responses.
L6 cells possess a higher number of IGF-1 receptor molecules than IR
molecules (52). Therefore, because of the presence of a high proportion
of IR/IGF-1R hybrid receptors, the reciprocal effects of insulin and
IGF-1 on PKB and MAPK were tested by prolonged incubation with insulin
or IGF-1 followed by a second exposure to either insulin or IGF-1. We
observed PKB and MAPK down-regulation after acute IGF-1 or insulin
treatment, irrespective of the agonist used in the 24-h pretreatment.
This suggests that heterologous desensitization occurs between insulin
and IGF-1. On the contrary, FCS could still activate MAPK after a
prolonged insulin treatment (Fig. 4C). These observations
imply that the PI3K/PKB and MAPK down-regulation depends on a signaling
event downstream of, but proximal to, the insulin/IGF-1 receptor.
Several post-receptor defects in insulin resistance have been reported,
including proteasome-mediated degradation of IRS-1, decreased IRS-2
mRNA levels, decreased GLUT4 protein expression levels (27, 30, 32,
53), as well as desensitization of IGF-1 and lysophosphatidic
acid-mediated MAPK activation via In 3T3-L1 adipocytes, proteasome-mediated IRS-1 degradation is
controlled by a rapamycin-sensitive pathway (46). On the contrary, in
our system as well as in Chinese hamster ovary/IR/IRS-1 cells (45),
insulin-induced IRS-1 degradation was not affected by rapamycin,
indicating that kinases upstream of mTOR, possibly PI3K itself, or
kinases independent of mTOR are required in L6 and Chinese hamster
ovary cells to elicit IRS-1 degradation. Similar to IRS-1, IRS-2 levels
were decreased upon prolonged insulin treatment in a
dose-dependent manner. In contrast to IRS-1
down-regulation, which was only dependent on PI3K, both LY294002 and
rapamycin blocked insulin-induced IRS-2 down-regulation. Furthermore,
exposure of starving cells to LY294002 or rapamycin induced an increase in the IRS-2 expression level (without affecting IRS-1 expression), thus a basal PI3K activity suffices to mediate IRS-2 down-regulation, via mTOR. Together, the above observations suggest that PI3K is the
chief molecule controlling insulin-induced degradation of both IRS-1
and IRS-2. Whereas insulin-dependent regulation of IRS-2
levels occurs in part by transcriptional repression of the IRS-2 gene
(56), we found that 32P labeling of insulin-treated L6
cells, in the presence of different inhibitors (LY294002, PD98059, or
rapamycin), resulted in a minimal 32P incorporation into
IRS-1, yet a higher tyrosine phosphorylation, only in LY294002-treated
cells. This indicates that the PI3K serine kinase activity directly
phosphorylates IRS-1 on serine/threonine residues, provoking its
degradation. In support of this is our observation that
immunoprecipitated IRS-1 from insulin-treated L6 is phosphorylated in
an in vitro kinase assay in a PI3K-dependent manner.2 On the contrary,
although mTOR inhibition caused approximately a 50% reduction in IRS-1
32P incorpozhyration, this was not sufficient to prevent
IRS-1 from being degraded, indicating that
mTOR-dependent phosphorylation sites do not
participate (or do not suffice for) in IRS-1 degradation.
We obtained further confirmation of the involvement of PI3K in IRS-1/2
down-regulation by expressing an agonist-independent, constitutively
active PI3K consisting of the catalytic subunit p110 To causally link insulin-induced down-regulation of PI3K/PKB and MAPK
pathways and degradation of IRS proteins, we exploited the possibility
of blocking insulin-induced IRS degradation by inhibiting PI3K. After a
24-h treatment with insulin in the presence of LY294002, IRS-1 and
IRS-2 expression levels were preserved. Subsequent insulin stimulation
(in freshly changed medium to remove LY294002 and recover PI3K
activation) restored PKB stimulation and greatly improved MAPK
responsiveness after the 102 nM insulin
treatment. We were unable to significantly regain MAPK activation after
103 nM insulin treatment. This is likely
because of the fact that at high concentrations insulin induces a
persistent dissociation of the Grb2·SOS complex, which can only be
reversed by insulin withdrawal (59) and might also explain the more
rapid kinetics of MAPK down-regulation as compared with PKB shown in
Fig. 2C. Having demonstrated the involvement IRS-1 and -2 down-regulation we next sought to determine the relative contribution
of each isoform by selective RNA interference knockdown of each
isoform. Whereas IRS-1 knockdown had no effect on the
insulin-stimulated activation of PKB and MAPK, we found that knockdown
of IRS-2 induced an almost total loss of insulin activation of PKB and
MAPK, thus defining IRS-2 as the chief molecule directing insulin
downstream signaling, at least in L6 cells.
In summary, our data indicate that prolonged insulin treatment of L6
muscle cells mimics the effects of hyperinsulinemia, leading to
down-regulation of both PI3K/PKB and MAPK signaling pathways and
glucose uptake via a decrease in IRS-1/2 docking molecules. We also
show that PI3K is the key molecule controlling the decrease in IRS-1/2
as down-regulation of IRS-1/2 is effectively prevented by PI3K
inhibition but induced by expression of a constitutively active PI3K.
Finally, we provide evidence for the existence of a causal link between
PI3K-elicited IRS-1/2 decrease and down-regulation of the PI3K/PKB and
MAPK pathways, with a prominent role played by IRS-2 as shown by RNAi
experiments. Our data suggest that, in L6 cells, insulin-induced
degradation of IRS molecules is driven by distinct mechanisms. Indeed,
for IRS-1 a PI3K-dependent pathway, with PI3K itself acting
as a IRS-1 serine/threonine kinase, appears to be involved, although
IRS-2 degradation is controlled by a PI3K-mTOR-dependent
mechanism. Moreover, tissue-specific mechanisms may control IRS
degradation, as IRS-1 degradation in adipocytes appears to be
mTOR-dependent (46) but mTOR-independent in L6 and Chinese
hamster ovary/IR/IRS cells (45). Because degradation of IRS proteins is
promoted by serine/threonine phosphorylation (21) and provides a
molecular link to insulin resistance (60), it will be of importance to
determine next the relative contribution of each serine/threonine
kinase among the several proposed, not the least PI3K itself, toward
IRS serine/threonine phosphorylation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells, leading to
hyperinsulinemia. In the long-term,
-cell failure to compensate for
peripheral insulin resistance leads to type 2 diabetes (1). Hyperinsulinemia, in association with increased circulating levels of
fatty acids (2), which exacerbate peripheral insulin resistance, is
thought to be a major factor contributing to progression to type 2 diabetes.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (> 5000 Ci/mmol), and
[32P]orthophosphate were from Amersham Biosciences
(Uppsala, Sweden). Recombinant human insulin was from Novo
Nordisk (Copenhagen, Denmark). IGF-1 and PDGF
/
were from
Calbiochem (La Jolla, CA). Secondary anti-mouse or anti-rabbit
antibodies conjugated to horseradish peroxidase were from Jackson
Laboratories (Copenhagen, Denmark). L-
-Phosphatidylinositol was from Sigma. TLC silica
plates were from Merck (Darmstadt, Germany). LY294002, rapamycin, and
PD98059 were from Calbiochem (La Jolla, CA) and TPCK was from Sigma.
Other chemicals were of the highest analytical grade available.
-subunit (Santa Cruz Biotechnology, Santa
Cruz, CA) antibodies. The proteins were detected by ECL after
incubation of the membranes with horseradish peroxidase-conjugated secondary antibodies. Signal intensities were measured with NIH Image
after scanning of non-saturated Eastman Kodak Biomax MR-1 films (Sigma).
-subunit, followed by
addition of protein A-Sepharose and further incubation for 2 h.
Immune complexes were washed twice with buffer A and then solubilized
in Laemmli sample buffer. If subjected to a PI3K assay, immune
complexes were washed twice with buffer A, twice with 0.1 M
Tris-HCl, 0.5 M LiCl, pH 7.4, and twice with lipid kinase
buffer (20 mM HEPES, 5 mM MgCl2, pH
7.4) prior to PI3K assay, which was performed on IRS-1
immunoprecipitates as described (26). 2-Deoxyglucose uptake was
measured as previously reported (34).
CAAX and IRS-1 were generated by
homologous recombination in Escherichia coli BJ 5183. Co-transformation of E. coli BJ 5183 led to recombination
between p110
CAAX (wild type and the kinase-dead K802R
(35)), IRS-1 (cloned in pCDNA3), and a viral vector recombinogenic
with the pCDNA3 cytomegalovirus promoter and poly(A) sequence
(VmcDNA, provided by S. Rusconi, University of Fribourg,
Switzerland) (36). Recombinants were screened by PCR analysis
with a pair of primers that annealed to the viral vector and
cytomegalovirus promoter sequence, respectively. A positive clone
harboring p110
CAAX (wt and KR) and IRS-1 was further
amplified in E. coli DH5
, digested with PacI,
and transfected by the calcium phosphate method into helper 293 cells
to produce viral particles. Adenoviruses were purified by
ultracentrifugation on CsCl gradient and stored in 0.1 M
Tris, 0.25 M NaCl, 1 mg/ml bovine serum albumin, 50% (v/v)
glycerol, pH 7.5 at
20 °C. Viral titer of stocks was
>108 plaque-forming units/ml.
-32P]dCTP using a random primer labeling kit
(Rediprime kit) and purified with a Probe Quant kit (both from Amersham
Biosciences). The probes encompassed the full-length coding regions of
GLUT1, PKB, and IRS-1. Hybridization was performed at 68 °C in
ExpressHyb hybridization solution (Clontech, Palo
Alto, CA), as per the manufacturer's instructions. Blots were
exposed for 24 h at
70 °C or visualized by phosphorimaging.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Insulin effect on PI3K and MAPK activities in
L6 myoblasts after prolonged exposure to insulin. L6 myoblasts
were treated with the indicated concentrations of insulin for 24 h. After 24 h, cells were exposed for 10 min to 1 µM
insulin (empty bars) or left untreated (solid
bars). A, top: IRS-1 associated PI3K
activity was measured in anti-IRS-1 immune complexes using
phosphatidylinositol as a substrate (see "Experimental
Procedures"; mean ± S.E., n = 3). PI3K activity
from cells not pretreated with insulin but exposed for 10 min to 1 µM insulin was taken as a 100%. Bottom,
representative anti-p85 immunoblot on IRS-1 immunoprecipitates
(IP) showing IRS-1-associated PI3K. B,
top: MAPK activation was evaluated by immunoblot analysis on
cell lysates with antibodies to active MAPK p42/p44. pMAPK signal from
cells not chronically treated and exposed for 10 min to 1 µM insulin was taken as a 100% (mean ± S.E.,
n = 3). Bottom, representative anti-pMAPK
and anti-MAPK immunoblots. WB, Western blot.
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Fig. 2.
Activation of PI3K downstream effector PKB,
kinetics of PKB and MAPK down-regulation and
[3H]2-deoxyglucose uptake after prolonged exposure to
insulin. L6 myoblasts were treated for 24 h with increasing
insulin concentrations as indicated. After 24 h, cells were
exposed for 10 min to 1 µM insulin. PKB activation was
evaluated by immunoblot analysis with antibodies to active PKB.
[3H]2-Deoxyglucose uptake was measured as described under
"Experimental Procedures." A, quantification of PKB
activation. Empty bars, 10 min exposure to 1 µM insulin; full bars, no second insulin
exposure (mean ± S.E., n = 3). PKB activation
from cells not chronically treated and exposed for 10 min to 1 µM insulin was taken as a 100%. B,
representative anti-pPKB immunoblot for the data shown in A. C, time course-dependent down-regulation of PKB
and MAPK. L6 myoblasts were exposed for 2, 6, and 18 h to
increasing insulin concentrations as indicated. Cells were then left untreated or exposed for 10 min
to 1 µM insulin. Time-dependent induction of
PKB and MAPK down-regulation was visualized by immunoblotting with
antibodies to pPKB Ser-473 and pMAPK. Representative immunoblots from
three independent experiments are shown. D,
[3H]2-deoxyglucose uptake after 10 min exposure to 1 µM insulin is expressed relative to cells not subjected
to 10 min exposure to insulin. (Mean ± S.E. is from three
independent experiments. In each independent experiment, triplicate
measurements yielded a S.E. < 6% of the mean values.)
2-fold increase in glucose uptake, as also shown with higher fold in
L6 myotubes (37), lead to a decreased insulin-induced [3H]2-deoxyglucose uptake following an acute insulin
treatment. This is shown by the ratio between the glucose uptake
measured with and without an acute insulin treatment following the 24-h chronic treatment (Fig. 2D).
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Fig. 3.
IR protein level decreases, but its relative
tyrosine phosphorylation level is not affected by prolonged insulin
treatment. L6 myoblasts were treated for 24 h plus an
additional 10 min as indicated. IR immunoprecipitates were analyzed for
total receptor content with an antibody to the insulin receptor
-subunit (upper blot) and for receptor phosphorylation
with antibodies to phosphotyrosine (lower blot). Molecular
masses are indicated on the right. Representative
immunoblots from four independent experiments are shown.
/
Pretreatment Does Not
Down-regulate Insulin Signaling--
L6 myoblasts express both insulin
and IGF-I receptors with a prevalence of IGF-1 receptors (40). Thus,
the insulin receptor mainly exists as a hybrid (
'
') with
the IGF-I receptor. To evaluate whether L6 myoblasts respond similarly
to a prolonged insulin or IGF-I pretreatment, cells were stimulated
with increasing concentrations of either insulin (0-103
nM range) or IGF-I (0-102 nM
range) for 24 h, followed by a 10-min treatment with either 1 µM insulin or 100 nM IGF-I. We observed a
down-regulation of PKB and MAPK after prolonged insulin treatment,
irrespective of the ligand used for the second exposure (insulin or
IGF-1, Fig. 4A). Likewise, a
24-h pretreatment with IGF-1 induced a desensitization of PKB and MAPK
when cells were subsequently exposed for a further 10 min to
either insulin or IGF-I (Fig. 4B). Given the presence of
insulin/IGF-1 hybrid receptors in L6 cells and the responsiveness of IGF-1R to insulin at high concentrations, the 24-h exposure to
insulin at the highest concentration (i.e. 103
nM) might lead to PKB and MAPK down-regulation by acting on
both receptors. Nevertheless, PKB and MAPK down-regulation was already occurring upon 24 h exposure to 10-102 nM
insulin (with no or little interference with IGF-1R activation) and to
1-10 nM IGF-1 (with no or little interference with IR
activation, Fig. 4, A and B). Given that (i)
down-regulation is already caused by exposure to low insulin/IGF-1
concentrations, and (ii) prolonged exposure to insulin diminishes acute
IGF-1 signaling and vice versa, we suggest that a down-regulation event
takes place at a postreceptor level.
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Fig. 4.
PKB and MAPK are activated by FCS and PDGF
but not by insulin/IGF-1 after 24 h of insulin/IGF-1 treatment,
although PDGF does not down-regulate insulin signaling. Anti-pPKB
and anti-pMAPK immunoblots are shown for the following experimental
conditions. A, following a 24-h insulin treatment as
indicated, L6 myoblasts were exposed for 10 min to either 1 µM insulin or 0.1 µM IGF-1. B,
following a 24-h IGF-1 treatment as indicated, L6 myoblasts were
exposed for 10 min to either 1 µM insulin or 0.1 µM IGF-1. C, top, L6 cells were
treated for 24 h with the indicated concentrations of insulin, and
then exposed for 10 min to either 1 µM insulin or 10%
(v/v) FCS. While it activated MAPK, FCS appeared to be ineffective for
PKB activation in L6 cells. Bottom, quantification of pMAPK
immunoreactivity. D, top: L6 cells were treated
with 10 nM PDGF for 24 h followed by a 10-min
stimulation with either 1 µM insulin (I) or 10 nM PDGF (P). Bottom, quantification
of pMAPK immunoreactivity. Representative immunoblots of at least two
independent experiments are shown (mean ± S.E. of pMAPK
immunoreactivity was of duplicate immunoblots from two independent
experiments). IP, immunoprecipitation; WB,
Western blot.
50%, respectively) after PDGF treatment, but no down-regulation of insulin signaling (Fig. 4D). This
indicates that down-regulation of insulin signaling strictly depends on a prior perturbation of the proximal insulin/IGF-1 receptor signaling pathway.
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Fig. 5.
24-h insulin treatment down-regulates
IRS-1/IRS-2 and up-regulates PKB expression. A, L6
myoblasts were treated for 24 h with increasing concentrations of
insulin as indicated. Total lysates were analyzed for IRS-1, IRS-2,
PKB, and MAPK expression. (The dots on the IRS-2 Western
blot indicate cross-reactive bands.) B, quantification of
the expression of PKB, IRS-1, and IRS-2, 100% refers to the expression
level of untreated cells (mean ± S.E., n 3).
C, Northern blot analysis of total RNA extracted from 24-h
insulin-treated L6. IRS-1 mRNA levels are similar, whereas PKB and
GLUT-1 mRNA levels are increased by insulin treatment in a
dose-dependent manner. Equal loading is visualized by
ethidium bromide staining of 18 S and 28 S rRNAs.
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Fig. 6.
Differential effects of LY294002, TPCK,
rapamycin, and PD98059 on IRS-1 and IRS-2 insulin-induced
down-regulation. A, L6 myoblasts were incubated for
24 h with increasing insulin concentrations in the presence of 50 µM LY294002 (+) or 0.1% (v/v) Me2SO ( ).
IRS-1 and IRS-2 expression levels were evaluated in total cell lysates
by immunoblottng with the respective antibodies. B,
immunoblotting with antibodies against pPKB confirmed the inhibitory
effect of LY294002 on the PI3K/PKB signaling pathway. Cells were
incubated for 24 h with insulin and LY294002 as indicated and
subjected to a further 10 min stimulation with 1 µM
insulin before lysis. C, L6 myoblasts were incubated for
24 h without or with 1 µM insulin in the presence of
0.1% (v/v) Me2SO (
), 5 µM or 17 µM TPCK, 50 µM LY294002 (LY), or
50 nM rapamycin (R) as indicated. The IRS-1
expression level was evaluated by immunoblotting with antibodies to
IRS-1. The inhibitory effect of TPCK, LY, and R was verified by band
shift analysis of p70 S6 kinase as well as by immunoblotting to pPKB.
Equal loading is demonstrated by immunoblotting to total PKB (performed
after strip of the immunoblot against the phosphorylated form).
D, L6 myoblasts were incubated for 24 h without or with
1 µM insulin in the presence of 0.1% (v/v)
Me2SO (
), 50 µM LY294002 (LY),
50 nM rapamycin (R), or 10 µM
PD98059 (PD) as indicated. IRS-1 and IRS-2 expression levels
were evaluated by immunoblotting of total lysates. The dots
indicate cross-reactive bands. The immunoblots shown are representative
of at least two independent experiments.
(Fig. 7B). Thus, although PI3K inhibition during a 24-h insulin treatment blocks
phosphatidylinositol 3,4,5-trisphosphate-mediated downstream signaling
in L6, the IRS-1·PI3K complex remains potentially active as
demonstrated by increased IRS-1/PI3K association, higher IRS-1 tyrosine
phosphorylation, and lower IRS-1 serine/threonine phosphorylation
level.
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Fig. 7.
In 24-h insulin-treated cells, LY294002
attenuates IRS-1 phosphorylation, increases IRS-1 phosphotyrosine
content, and increases IRS-1 associated p85 .
A, left: IRS-1 was immunoprecipitated from
32P-labeled L6 myoblasts treated for 24 h with insulin
in the presence of 0.1% (v/v) Me2SO (
), 50 µM LY294002 (LY), 50 nM rapamycin
(R), or 10 µM PD98059 (PD) as
indicated. The IRS-1 associated radioactivity was visualized by
phosphorimaging after separation with a 7.5% SDS-PAGE.
Right, IRS-1 relative phosphorylation was calculated by
dividing the incorporated radioactivity by the IRS-1 expression level
(C,
-IRS-1 immunoblot, lanes 5-8). A result
representative of two independent experiments is shown. B,
IRS-1 was immunoprecipitated from L6 myoblasts treated with insulin in
the presence of 0.1% (v/v) Me2SO (
), 50 µM
LY294002 (LY), 50 nM rapamycin (R),
10 µM PD98059 (PD), 200 nM GF109
(GF), or 10 µM MG132 (MG) as
indicated. IRS-1 tyrosine phosphorylation (upper blot) and
IRS-1-associated PI3K adapter p85
(lower blot) are shown.
A representative blot of four independent experiments is shown.
CAAX wt) or, as a control, a kinase-dead mutant
(p110
CAAX KR, Fig.
8A). Expression of p110
CAAX wt (but not KR) activated the PI3K downstream targets
PKB (Fig. 8B) and p70 S6 kinase (not shown) (50), and
induced a decrease in IRS-1 and IRS-2 protein expression, thus
confirming that IRS-1/2 down-regulation can be induced in a
receptor-independent manner and is controlled by PI3K.
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Fig. 8.
Expression of a catalytically active
p110 CAAX down-regulates
IRS-1 and IRS-2 proteins. A, L6 myoblasts were infected
with adenovirus expressing p110
CAAX wt and kinase dead
(KR). Transcription of the p110
CAAX mRNA
was visualized by Northern blot. B, L6 myoblasts were
incubated overnight in DMEM without serum and with increasing
multiplicity of infection of adenovirus expressing p110
CAAX wild type (wt) or kinase-dead. The cells
were then kept in DMEM, 10% FCS (v/v) for 36 h followed by
overnight starvation prior to lysis and immunoblotting with antibodies
to IRS-1, IRS-2, and active PKB (phospho-Ser-473) as indicated. The
blots shown are representative of two independent experiments.
C, quantification of IRS-1 and IRS-2 expression levels in
uninfected cells and infected cells infected at a m.o.i. of 100 (mean ± S.E., n = 3).
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Fig. 9.
Inhibition of insulin-induced IRS-1 and IRS-2
down-regulation by LY294002 reverses the inhibitory action of a
prolonged insulin treatment on PKB and MAPK activation. L6
myoblasts were incubated for 24 h in the presence of 0, 102, and 103 nM insulin and 50 µM LY294002 as indicated. After 24 h incubation,
cells were left untreated or were treated for 10, 30, or 60 min with 1 µM insulin. Prior to exposure to insulin, LY294002
containing medium was replaced with fresh DMEM to allow PI3K/PKB
activation. Cell lysates were then subjected to immunoblotting with
antibodies against active PKB, active MAPK, IRS-1 and IRS-2. Cell
lysates were then subjected to immunoblotting with antibodies against
active PKB, active MAPK, IRS-1 and IRS-2.
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Fig. 10.
IRS-2 is the adapter accounting for
insulin-induced activation of PKB and MAPK in L6 cells. IRS-1,
IRS-2, or both were knocked down by RNA interference. RNAi was
performed by calcium phosphate transfection of dsRNAs. 24 h
post-transfection, cells were starved overnight and treated or not for
10 min with 1 µM insulin as indicated prior to cell
lysis. A, cell lysates were then subjected to immunoblotting
with antibodies to IRS-1, IRS-2, PKB Ser-473, and active MAPK as
indicated. Equal loading is demonstrated by immuno- blotting to total MAPK, total PKB (performed after stripping the
immunoblot against the phosphorylated form), and p85 PI3K adapter
subunit. The blots shown are representative of at least four
independent ones. B, quantification of the expression level
of IRS-1 (left graph) and IRS-2 (right graph)
following RNAi to IRS-1, IRS-2, and IRS-1 + IRS-2. Expression levels
with no addition of siRNA is taken as 100% (mean ± S.E.,
n = 4). C, to assess the relevance of the
partial decrease of IRS-1 following RNAi to IRS-2, after knockdown of
IRS-2 cells were infected by an adenovirus expressing IRS-1 (Ad
IRS-1) at the indicated multiplicity of infection. After overnight
starvation, cells were left untreated or treated with 1 µM insulin for 10 min. Immunoblotting with antibodies to
IRS-1, IRS-2, PKB phospho-Ser-473, and active MAPK was performed as
indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestin-1 down-regulation (54).
Thus, we tested whether the insulin-induced insulin signaling
down-regulation might depend on alterations in the levels of IRS
molecules and/or the downstream kinases PKB and MAPK. We observed an
insulin-induced down-regulation of IRS protein levels that parallels
the down-regulation of PI3K/PKB and MAPK. IRS-1 down-regulation could
be blocked by PI3K inhibition with LY294002, but not by PDK1, mTOR, or
MAPK inhibition. Of particular interest is the observation that
insulin-induced IRS-1 degradation was not blocked by TPCK, an inhibitor
of PDK1 action. As PI3K-activated PDK1 phosphorylates and activates
several downstream kinases, including PKB, p70 S6 kinase, SGK, RSK, and
atypical protein kinase Cs (55), the involvement of these kinases in
IRS-1 phosphorylation in L6 cells can be excluded.
fused at its C
terminus to the farnesylation signal of H-Ras. This moiety targets the
protein to the plasma membrane (p110
CAAX), activating it
(57). In the absence of insulin, expression of p110
CAAX
wild type (but not kinase-dead p110
CAAX K802R) was
sufficient to activate PKB, in a multiplicity of
infection-dependent manner and induced a decrease in both
IRS-1 and IRS-2 as observed in 3T3-L1 adipocytes (58).
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ACKNOWLEDGEMENTS |
---|
We thank J. Downward for the
pcDNA3-myc-p110-CAAX construct, B. Hemmings for
antibodies to PKB, and S. Rusconi for the adenoviral vector
VmAdCDNA. We thank S. Longnus for critically reading the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by INSERM, Université de Nice-Sophia-Antipolis, la Région PACA, European Community Grant QLGI-CT-1999-00674, EuroDiabetesGene, and QLK3-CT-2000-01038, and Aventis Pharma, Frankfurt, Germany, contract 99206.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.
Supported by an INSERM "Poste Vert" postdoctoral fellowship
and in part by the Fondation pour la Recherche Médicale.
§ Supported by European Community Grant QLGI-CT-1999-00674.
¶ Supported by INSERM "Poste Vert" postdoctoral fellowships.
To whom correspondence should be addressed. E-mail:
vanobbeg@unice.fr.
Published, JBC Papers in Press, February 18, 2003, DOI 10.1074/jbc.M208984200
2 L. Pirola and S. Bonnafous, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
IR, insulin
receptor;
IRS, insulin receptor substrate;
PI3K, phosphoinositide
3-kinase;
TPCK, N-tosyl-L-phenylalanyl
chloromethyl ketone;
MAPK, mitogen-activated protein kinase;
PKB, protein kinase B;
IGF-1, insulin-like growth factor 1;
PDGF, platelet-derived growth factor;
DMEM, Dulbecco's modified Eagle's
medium;
FCS, fetal calf serum;
ds, double stranded;
RNAi, RNA
interference;
siRNA, short interfering double stranded RNA;
m.o.i., multiplicity of infection.
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