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Original Studies |
INSERM Unité 402, Faculté de Médecine Saint-Antoine (M.A., C.V., C.D.-M., O.L., G.C., J.C., M.C.), 75571 Paris Cedex 12; Service de Biochimie, Hôpital Rothschild (C.V., J.C.), 75571 Paris Cedex 12; and Département de Médecine J, Hôpital Brabois (J.D., P.K.), 54500, Vandoeuvre les Nancy, France
Address all correspondence and requests for reprints to: Dr. Martine Caron, INSERM Unit 402, Faculté de Médecine Saint-Antoine, 27 rue Chaligny, 75571 Paris Cedex 12, France. E-mail: caron{at}st-antoine.inserm.fr
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The type B syndrome of extreme insulin resistance is a rare clinical
disorder characterized by the presence of circulating autoantibodies
directed against the IR (AIRs) (10, 11). It is usually associated with
hyperinsulinemia, hyperglycemia, and acanthosis nigricans. Most AIRs
are polyclonal IgGs that compete with insulin for binding to the
-subunit of the IR. Although in some cases autoantibodies from type
B patients were shown to be directed against an antigenic site found in
both IR and IGF-IR or to contain subpopulations of antibodies directed
against each receptor (12, 13), in most cases AIRs are specific of the
IR. In cultured cells, AIRs display both short term insulin-like
properties (14) and long term antagonizing effects on insulin action
(15, 16, 17), the latter being associated in some cases with a progressive
loss of cellular IRs (18, 19, 20). To our knowledge, the effect of AIRs on
IGF-I signaling has never been studied.
In the present study, we report the case of a 68-yr-old woman with clinical signs of autoimmune disease and extreme insulin resistance due to AIRs. In short term experiments, patient AIRs blocked insulin binding and mimicked insulin action in cultured CHO cells overexpressing human IRs, but failed to alter [125I]IGF-I binding. Most important, when used in long term experiments, patient AIRs induced severe cell resistance not only to insulin, but also to IGF-I, although they affected neither the IGF-IR expression level nor IGF-IR autophosphorylation. The mechanisms underlying this molecular resistance were investigated. Our results provide the first evidence that AIR-desensitized IRs exerted a dominant negative effect on IGF-I signaling at a step distal to IGF-IRs through a mechanism involving the constitutive association of the IRs with IRS-1 and IRS-2.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The patient, a 68-yr-old French woman, developed in about 3
months many clinical and biological symptoms indicative of immune
dysfunction. Several criteria allowed the diagnosis of systemic lupus
erythematosus with renal involvement. She was treated with prednisone
(0.5 mg/kg·day) and an iv bolus of cyclophosphamide (500
mg/m2·month). This treatment did not improve her clinical
state, and 3 months later she displayed massive polyuria and polydipsia
due to a severe insulin-resistant nonketotic diabetes. Despite iv
insulin treatment (6 IU/kg·day), hyperglycemia remained elevated
(
25 mmol/L), and in a few weeks the patient developed histologically
proven acanthosis nigricans. As described below, AIRs were found in the
patient’s serum that defined a type B syndrome of insulin resistance.
At this time, angioimmunoblastic lymphoadenopathy with dysproteinemia
was diagnosed on the basis of typical histological findings from a
cervical node. A high dose of prednisone (2 mg/kg·day) was required
to improve the clinical and biological features of autoimmunity. Within
2 weeks, the titer of AIRs decreased by about 6-fold, and 10 days later
insulin treatment was no longer required. At that time, circulating
AIRs were not detected. Three months later the patient was in remission
with normal glucose tolerance.
Chinese hamster ovary (CHO) cells
Untransfected CHO cells expressing 2 x 103 endogenous IRs and 4 x 104 endogenous IGF-IRs and CHO cells expressing 5 x 105 human IRs (CHO-IR cells) were cultured as previously described (21). For insulin and IGF-I stimulation, porcine insulin (Novo, Copenhagen, Denmark) or recombinant human IGF-I (PreproTech, London, UK) was added at 50 or 100 nmol/L as indicated. Binding of insulin or IGF-I was performed in the presence of either 19 pmol/L [125I]insulin (3-[125I]iodotyrosylA14 insulin; 2000 Ci/mmol; Amersham Pharmacia Biotech France SA, Les Ulis, France) or 30 pmol/L [125I]IGF-I (3-[125I]iodotyrosyl IGF-I; 2000 Ci/mmol; Amersham Pharmacia Biotech France SA) following standard procedures (22).
Characterization of AIRs in the patient’s serum
Patient and control serum samples were used either directly or after treatment with 33% ammonium sulfate at 4 C for 4 h to obtain IgG-enriched fractions (17). Increasing dilutions of patient serum collected when hyperglycemia was maximal inhibited the binding of [125I]insulin to CHO-IR cells (20.9 ± 4.0 fmol/mg protein), with a half-maximal effect at a 1:500 dilution. The inhibitory activities were retained in the IgG fraction of the patient’s serum, with maximal and half-maximal effects at 250 and 15 µg/mL, respectively. Patient serum samples collected after normalization of glucose tolerance as well as the related IgGs did not inhibit insulin binding. Patient IgGs did not modify the ability of [125I]IGF-I to bind the IGF-IR at 22 C for 90 min (4.90 ± 1.05 and 4.34 ± 9.08 fmol/mg protein in CHO-IR cells preincubated with control and patient IgGs, respectively). These results were consistent with the presence in the patient serum of autoantibodies directed against the IR, but not against the IGF-IR.
Cell desensitization by long term treatment with patient AIRs
The ability of long term treatment with AIRs to induce cell resistance to insulin and IGF-I was evaluated by incubating serum-free CHO-IR cells with a maximally effective concentration of patient IgGs (250 µg/mL) for 8–16 h at 37 C. After two washes with ice-cold PBS, AIRs were dissociated from the cell surface by a 3-min acid wash at 4 C in a buffer containing 100 mmol/L HEPES, 120 mmol/L NaCl, 1.2 mmol/L MgSO4, 5 mmol/L KCl, and 15 mmol/L sodium acetate, pH 3.5, as described previously (17). Washed cells were then tested for their ability to respond to insulin and IGF-I for various metabolic and mitogenic processes, as described below.
The acid wash altered neither cell integrity nor sensitivity to insulin and IGF-I, as evaluated by cell number, protein content, [125I]insulin binding capacity, and insulin and IGF-I stimulation of IRS-1 tyrosine phosphorylation and MAPK activity, which were similar in unwashed and acid-washed cells. The efficiency of the acid wash to dissociate AIRs from the cell surface was assessed by verifying that 1) the lysates from AIR-treated cells that were subjected to the acid wash did not contain immunoreactive IgGs; immunodetection was performed by blotting Protein A/G Plus-agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) immunoprecipitates with protein A/horseradish peroxidase (Transduction Laboratories); and 2) the acid wash reversed the short term inhibition of insulin binding induced by AIRs; this was shown by measuring [125I]insulin binding at 15 C for 5 h in CHO cells treated with patient IgGs for 1 h at 22 C that were subjected, or not, to the acid wash.
Glycogen and DNA syntheses
Cell monolayers (5 x 105 cells) were incubated for 2 h with insulin or IGF-I and 2 µCi/mL D-[U-14C]glucose (300 mCi/mmol; Amersham Pharmacia Biotech). Glycogen was then extracted as described previously (6). The results are given as a percentage of the basal value, which is expressed as picomoles of [14C]glucose incorporated into glycogen per mg protein/h.
Subconfluent CHO-IR cells or cultured human fibroblasts (105 cells) were maintained for 24 h in serum-free DMEM, treated or not with IgGs for 8 h, and subjected to the acid wash. Cells were then incubated for 16 h with the indicated ligand followed by the addition of 0.5 µCi [methyl-3H]thymidine (5 Ci/mmol; Amersham Pharmacia Biotech) for 4 h. DNA was extracted as described previously (6). The results are given as a percentage of basal value, which is expressed as disintegrations per min/mg protein.
Western blotting studies
Cell monolayers (2 x 106 cells) were lysed at
4 C for 30 min in a buffer containing 50 mmol/L HEPES, 50 mmol/L NaF,
100 mmol/L NaCl, 5 mmol/L ethylenediamine tetraacetate (EDTA), 5 mmol/L
ethyleneglycol-bis-(ß-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid, 0.2 mmol/L Na3VO4, 1 µg/mL leupeptin,
0.2 mmol/L PMSF, and 1% Triton X-100, pH 7.4. The lysate was clarified
by centrifugation, and equal protein amounts (500 µg) were incubated
with the indicated antibody for 2 h at 4 C. Protein A/G
Plus-agarose (25 µL) was then added for 16 h. Aliquots of the
immunoprecipitates (corresponding to 150 µg cell lysate) were washed
three times in lysis buffer containing 0.1% Triton X-100 and processed
for SDS-PAGE and Western blotting. Immune complexes were visualized by
chemiluminescence (enhanced chemiluminescence detection kit,
Amersham Pharmacia Biotech). The antibodies used in these
study were obtained from Santa Cruz Biotechnology, Inc.
(insulin Rß C-19, IGF-I R 2C8, IGF-I Rß C-20, IRS-1 C-20),
Upstate Biotechnology, Inc. (Lake Placid, NY; IRS-2
06–506), and Transduction Laboratories (Lexington, KY;
antiphosphotyrosine antibody RC20H). When indicated, cell monolayers
(7 x 105 cells) were solubilized in Laemmli sample
buffer containing 100 mmol/L dithiothreitol, and aliquots of whole cell
lysates (8 µg) were processed for SDS-PAGE and Western blotting.
MAPK assay
Cell monolayers (7 x 105 cells) were lysed in a buffer containing 50 mmol/L Tris, 50 mmol/L NaF, 100 mmol/L NaCl, 5 mmol/L EDTA, 40 µmol/L ß-glycerophosphate, 0.2 mmol/L Na3VO4, 1 µg/mL leupeptin, 0.2 mmol/L PMSF, and 1% Triton X-100, pH 7.5. Aliquots of cell lysates (8 µg) were subjected to SDS-PAGE and Western blotting with an antibody directed against the activated forms of extracellularly regulated kinases (anti-active MAPK polyclonal antibody, Promega Corp., Madison, WI).
PI 3-K assay
CHO-IR cells (2 x 106 cells) were lysed for 20
min at 4 C in a buffer containing 20 mmol/L Tris, 0.137 mmol/L NaCl, 1
mmol/L MgCl2, 1 mmol/L CaCl2, 0.1 mmol/L
Na3VO4, 1% Nonidet P-40, 10% glycerol, and 1
mmol/L PMSF, pH 8.0. Lysates were clarified by centrifugation, and the
supernatants (500 µg) were immunoprecipitated with an anti-PY
antibody (PY 99, Santa Cruz Biotechnology, Inc.) in the
presence of 25 µL Protein A/G Plus-agarose. After successive
washes at 4 C with PBS; 100 mmol/L Tris, 500 mmol/L LiCl (pH 7.4); 10
mmol/L Tris, 100 mmol/L NaCl, and 1 mmol/L EDTA (pH 7.4), pellets were
resuspended in the latter buffer (final volume, 50 µL) containing 1
mmol/L MgCl2, 20 µg L--phosphatidylinositol (sonicated
in 5 mmol/L HEPES, pH 7.4), 50 µmol/L ATP, and 12 µCi
[
-32P]ATP (2000 Ci/mmol; Amersham Pharmacia Biotech). The reaction was stopped 30 min later by the addition
of 100 µL 1 mol/L HCl and 200 µL CHCl3/methanol (1:1).
Samples were centrifuged, and the lower organic phase was recovered,
washed with 80 µL HCl/methanol (1:1), and applied to a silica gel TLC
plate (Merck KGaA, Darmstadt, Germany) coated with 1% potassium
oxalate. TLC plates were developed in
CHCl3/CH3OH/H2O/NH4OH
(60:47:11.3:2) and dried, and labeled lipids were visualized by
autoradiography. Standard lipids were run in parallel and colored with
iodine vapor.
Data analysis
Chemiluminescent and radioactive signals were quantified by scanning densitometry using NIH Image 1.5 software. Results are expressed as the mean ± SEM for the indicated number of experiments.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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As a result of AIR interaction with the IR, we observed short term
stimulation of several insulin-related events (Fig. 1
). Patient IgGs (lanes 4) enhanced IR
ß-subunit and IRS-1 tyrosine phosphorylation (Fig. 1A) and increased
the level of activated ERK1 and ERK2 (Fig. 1B) to the same extent as
did a maximally effective concentration of insulin (lanes 2). In
contrast, control IgGs were ineffective (lanes 3). The stimulations by
patient IgGs were dose dependent and specific, as they were abolished
by 30-min pretreatment with a 1:25 dilution of anti-human IgG serum
(data not shown). The maximal effects of insulin and patient IgGs were
not additive (lanes 5).
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The next set of experiments was designed 1) to examine the ability
of patient AIRs to induce insulin desensitization in vitro
in cultured CHO cells and 2) to check whether these AIRs could also
induce cell resistance to IGF-I, a hormone that shares with insulin
several biological activities (1, 2). As shown in Table 1, long term treatment of CHO-IR cells
with patient IgGs (250 µg/mL) severely decreased insulin and IGF-I
(50 nmol/L) stimulation of glycogen synthesis compared to that in
untreated cells without significantly modifying the basal level. The
effect of patient IgGs was specific, as treatment of the cells with
control IgGs (300 µg/mL) did not modify the ability of insulin and
IGF-I to stimulate glycogen synthesis. AIR-treated cells also failed to
respond to insulin and IGF-I (100 nmol/L) for the stimulation of DNA
synthesis. It must be noted that long term treatment with AIRs weakly
increased basal DNA synthesis, an effect that was also observed in
cells preexposed to control IgGs.
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), two kinases involved in the
stimulation of glycogen and DNA syntheses. Indeed, the stimulation of
PI 3-K by insulin or IGF-I (50 nmol/L) was observed in cells treated
with control IgGs (Fig. 2A, lanes 5 and 6), but was lost in cells
chronically treated with patient IgGs (lanes 8 and 9), which displayed
a slight increase in basal PI 3-K activity. Patient AIRs also caused
cell resistance to insulin and IGF-I for the activation of MAPK, as
evaluated by Western blot analysis with an antibody specific for the
phosphorylated forms of ERK1 and ERK2 (Fig. 2B, compare lanes 8 and 9
with lanes 2 and 3 or 5 and 6). Taken together these findings showed
that patient AIRs could induce severe insulin resistance in CHO-IR
cells, and that this defect was associated with a severe resistance of
the cells to IGF-I.
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To explore the mechanisms involved in cell resistance to insulin
and IGF-I induced by patient AIRs, we searched for possible defects at
the IR and IGF-IR levels. Long term treatment of CHO-IR cells with
patient IgGs (250 µg/mL) decreased the IR expression level by 50%,
as detected by immunoblotting anti-IR immunoprecipitates with an
anti-IR ß-subunit antibody (Fig. 3A, left panel, lane 3). In accordance, equilibrium binding
studies performed at 15 C for 5 h indicated that
[125I]insulin binding was 50% lower in AIR-treated cells
than in untreated cells (9.75 ± 0.86 and 20.3 ± 1.6 fmol/mg
protein, respectively); this was confirmed by Scatchard analysis of the
data, which indicated a decreased IR number without an appreciable
change in IR affinity (not shown). These results are consistent with
previous studies showing that long term treatment with AIRs could
induce IR down-regulation (18, 19, 20). In contrast, cell treatment with
patient AIRs altered neither the level of immunoreactive IGF-IRs
(Fig. 3A, right panel, lane 3) nor the binding of
[125I]IGF-I to CHO-IR cells (6.25 ± 1.31 and
5.92 ± 1.38 fmol/mg protein in control and patient IgG-treated
cells, respectively).
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). This was observed in anti-IR ß-subunit
(left panel, lane 6) and anti-PY (right panel,
lane 4) immunoprecipitates. However, patient IgGs failed to modify the
stimulatory effect of IGF-I on IGF-IR autophosphorylation (Fig. 3C)
regardless of whether detection was performed after immunoprecipitation
of cell lysates with an anti-IGF-IR -subunit (left panel,
lane 4) or an anti-PY antibody (right panel, lane 4). Taken as a whole, the results showed that patient AIRs caused cell resistance to insulin signaling at the IR level by two mechanisms: down-regulation of IR number and loss of activation of IR autophosphorylation. In contrast, AIRs failed to modify the expression level of IGF-IRs or their ability to autophosphorylate in response to IGF-I, indicating that they induced cell resistance to IGF-I at a step distal to IGF-I-R activation.
Long term treatment with patient AIRs blocked insulin and IGF-I stimulation of IRS-1 and IRS-2 tyrosine phosphorylation and caused IRS-1 and IRS-2 constitutive association with IRs
An early postreceptor step shared by both IRs and IGF-IRs is
tyrosine phosphorylation of IRS-1 (2, 3). We thus tested whether AIRs
modified IRS-1 tyrosine phosphorylation in response to these hormones
(Fig. 4A). We initially observed by
immunoblotting anti-IRS-1 immunoprecipitates with an anti-PY antibody
that desensitized IRs did not mediate insulin stimulation of IRS-1
tyrosine phosphorylation (lane 8). The AIR-induced defect was specific,
as it could not be produced by control IgGs (lane 5), and it could not
result from a decreased level of IRS-1 protein (Fig. 4B, lanes 7–9).
Alternatively, despite the ability of IGF-IRs to autophosphorylate
in response to IGF-I (Fig. 3C), they could not transduce IRS-1 tyrosine
phosphorylation in cells treated with patient IgGs (Fig. 4A, lane 9).
As overexpression of human IRs in CHO cells can lead to the formation
of insulin/IGF-I receptor hybrids (23), it was important to
determine whether the defect in IGF-I action induced by AIRs could
be due to the presence of such hybrids in CHO-IR cells. To examine this
issue, the above experiments were repeated in parental CHO cells (Fig. 4C), and again, we observed resistance to IGF-I (and insulin) in cells
treated with patient IgGs (lanes 6 and 5) compared to that in cells
treated with control IgGs (lanes 3 and 2). These results strongly
suggested that the blockade of IGF-I signaling induced by patient AIRs
occurred at an early postreceptor step, distal to IGF-R phosphorylation
but proximal to IRS-1 phosphorylation, by a mechanism that presumably
involved a defect in IRS-1 assembly with IGF-IRs.
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), we performed coimmunoprecipitation
assays with an anti-IRS-1 (left panel) and an anti-IGF-IR
-subunit (right panel) antibody. We observed that in
CHO-IR cells treated with control IgGs, IGF-IRs were complexed with
IRS-1 20 min after IGF-I addition (lanes 2) under conditions where
maximal tyrosine phosphorylation of both IGF-IR (Fig. 3C, lanes 2) and
IRS-1 (Fig. 4A, lane 6) occurred (24). In contrast, IGF-IR.IRS-1
complexes were hardly detected in AIR-treated cells stimulated with
IGF-I (Fig. 5A, lanes 4), suggesting that the defect of IGF-I signaling
resulted from the inability of activated IGF-IRs to form a
complex with IRS-1 in AIR-treated cells.
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)
with an anti-IRS-1 (left panel) or an anti-IR ß-subunit
(right panel) antibody indicated that the association of IR
with IRS-1 was strictly different in AIR-treated cells (left
panel, lanes 5 and 6, and right panel, lanes 3 and 4)
and in cells treated or not with control IgGs (left panel,
lanes 1 and 2, and lanes 3 and 4) regardless of whether they were
stimulated with insulin for 20 min. In fact, AIR treatment profoundly
altered the kinetics of IR.IRS-1 association (Fig. 5C); in control
cells stimulated by insulin (left panel), IR and IRS-1
formed a transient complex that was observed at 2 min, persisted up for
10 min (not shown), and disappeared at 20 min, a time at which IR (Fig. 3B, lane 2) and IRS-1 (Fig. 4A, lane 2) tyrosine phosphorylation was
maximal. These results were consistent with previous studies that
showed that phosphorylated IRS-1 rapidly dissociates from the IR after
insulin stimulation (3, 25, 26, 27). In contrast, in AIR-treated cells
(Fig. 5C, right panel), IR-IRS-1 complexes were formed
constitutively in the absence of insulin and were unmodified by the
hormone. In these cells, sequestration of IRS-1 by the IR ß-subunit
correlated with impaired IR (Fig. 3B, lane 6) and IRS-1 (Fig. 4A, lane
8) tyrosine phosphorylation. Similar assays conducted in parental CHO
cells (Fig. 5D) showed that AIRs also induced constitutive association
of IRs with IRS-1 in untransfected cells (lane 4) regardless of whether
insulin (lane 5) and IGF-I (lane 6) were present. This rules out the
possibility that the AIR effect seen in CHO-IR cells reflected a forced
mechanism due to IR overexpression. To check whether the bulk of IRS-1
was sequestered by IRs in AIR-treated cells (Fig. 5E), supernatants
from either the anti-IR ß-subunit (right panel) or the
anti-IRS-1 (left panel) immunoprecipitate were subjected to
a second immunoprecipitation with either an anti-IRS-1 or an anti-IR
ß-subunit antibody. The results indicated the absence of
immunoreactive IRS-1 (right panel, lanes 3 and 4) or IR
ß-subunit (left panel, lanes 3 and 4) in the supernatants
of the anti-IR ß-subunit and the anti-IRS-1 immunoprecipitates from
cells treated with patient IgGs.
As IRS-2, the alternate substrate of IRS-1 (28, 29), could compensate
for disrupted insulin and IGF-I signaling through IRS-1 in AIR-treated
cells, we repeated the above experiments with an anti-IRS-2 antibody
(Fig. 6). We observed that insulin and
IGF-I also failed to increase IRS-2 tyrosine phosphorylation in
AIR-treated cells (Fig. 6A, left panel, lanes 4–6), which
could not be explained by a decreased level of IRS-2 protein
(right panel, lanes 5 and 6), but correlated with
sequestration of the substrate by desensitized IRs (Fig. 6B, left and right panels, lanes 3 and 4).
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Patient AIRs failed to block the effects of agents that activate MAPK and mitogenesis independently of IRS-1
The results described above showed that treatment of CHO-IR cells
with patient AIRs inhibited the ability of insulin and IGF-I to
increase tyrosine phosphorylation of IRS-1 and IRS-2 and to activate
MAPK and DNA synthesis. These inhibitory effects of the AIRs could be
linked or not. To distinguish between these possibilities, we examined
whether the MAPK pathway remained functional in AIR-treated cells by
using vanadyl sulfate (VS) and FCS, which activate these kinases
through a pathway independent of IRS-1. Indeed, Fig. 7A shows that VS (100 µmol/L) and FCS
(10%) markedly enhanced MAPK activation in CHO-IR cells treated with
control IgGs (lanes 3 and 4), whereas they did not increase IR
ß-subunit and IRS-1 tyrosine phosphorylation (Fig. 7B, lanes 3 and
4). Whereas cell treatment with patient IgGs almost blocked insulin
activation of MAPK (Fig. 7A, lane 6), it failed to modify the VS- or
FCS-mediated activation of MAPK in these cells (lanes 7 and 8).
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shows that whereas the action of insulin and IGF-I on DNA synthesis was
severely impaired in cultured fibroblasts pretreated with patient IgGs,
the stimulatory effect of FCS, EGF, and PDGF was unchanged.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In contrast, the hypothesis invoking the substrate competition to account for the blocking of IGF-I signaling in AIR-treated cells provides an explanation for our results as developed below. Coimmunoprecipitation assays with antibodies directed against IR, IGF-IR, and IRS-1 showed that in AIR-treated cells, the bulk of cellular IRS-1 was constitutively sequestered in a stable complex by desensitized IRs, which prevented IRS-1 association with ligand-activated IGF-IRs. Sequestration of IRS-1 by kinase-defective IRs was reported in previous studies (25, 34) and was suggested to result from altered conformation of the IR tyrosine kinase domain. From crystallographic data, a model for IRS-1 assembly with the IR was proposed (27, 35) in which insulin binding to control cells alters the spatial conformation of the IR and promotes IR autophosphorylation. This allows unrestricted access of IRS-1 to the IR substrate-binding pocket and IRS-1 binding to Tyr960 in the juxtamembrane of the IR through its phosphotyrosine binding domain (3). Bound IRS-1 is then tyrosine phosphorylated by the activated IR kinase, and once SH2-proteins are engaged with IRS-1, it dissociates from the IR. In light of this model, a possible explanation for the present data is that the persistent anchoring of AIRs at or in a close proximity to the insulin-binding site disrupted the spatial conformation of the IRs so that they were stabilized in a pseudoactivated conformation, with an opened substrate-binding pocket easily accessible for IRS-1. However, due to defective kinase activity of desensitized IRs, bound IRS-1 could not be phosphorylated in response to insulin, remaining stably associated with the IRs and hence unavailable for engagement with activated IGF-IRs.
Our investigations also showed that AIRs induced sequestration of IRS-2 by the IRs and defective IRS-2 phosphorylation in response to insulin or IGF-I. This finding may be surprising, because it was shown that, in contrast to IRS-1, which can associate with unphosphorylated IRs (25, 36), IRS-2 only associates with tyrosine-phosphorylated IRs (37). However it must be underlined that IRs from AIR-treated cells exhibited a low level of tyrosine phosphorylation, which could presumably be sufficient to allow IRS-2 (and presumably IRS-1) engagement by the IRs, but not to promote their phosphorylation, an event that is essential for IRS dissociation from the IRs (3, 37). It is thus possible that IRS-1 and IRS-2 are stably linked to the IRs by a mechanism involving Tyr960 of the IR. Taken as a whole, these data show that IRS-1 and IRS-2 are sequestered by desensitized IRs and that none of them could transduce the insulin signal or serve as a substrate for ligand-activated IGF-IRs in AIR-treated cells.
Interestingly, AIRs also blunted insulin and IGF-I activation of MAPK and DNA syntheses, which are classically initiated by the engagement and tyrosine phosphorylation of Shc (24, 38, 39, 40). Although both IRS-1 and Shc bound Tyr960 of the IR through their phosphotyrosine binding domain, their time course of association and their mode of interaction are different (41). IRS-1 binds Tyr960 regardless of whether it is phosphorylated (36, 42), whereas Shc binding is slower and requires phosphorylation of both Tyr960 in the juxtamembrane domain and Tyr1316 and Tyr1322 in the carboxyl-terminal domain of the IR (36, 41, 43). Thus, Shc signaling of MAPK and DNA synthesis in response to insulin and IGF-I is presumably inhibited in AIR-treated cells due to the misactivation and engagement of the IRs in stable complexes with IRS proteins.
The failure of IGF-I to signal MAPK activation and mitogenesis in AIR-treated cells may suggest that kinase-competent IGF-IRs are unable to engage Shc. However, the relative contributions of Shc and IRS-1 in the activation of the MAPK signaling cascade are complex and not clearly defined (3, 44). MAPK activation may also be initiated by the engagement of IRS-1 with PI 3-K (45) or Grb-2-SOS (46, 47) or by the formation of a large signaling complex including IRS-1 and Shc (38, 48, 49). In light of these data, the loss of MAPK activation by IGF-I in AIR-treated cells may be explained by the inability of IRS-1 to associate with Grb-2-SOS, PI 3-K, or Shc. Consistent with this explanation, we provide evidence that AIR treatment affected neither the stimulation of MAPK and/or mitogenesis by vanadate, FCS, and PDGF, all of which act through an IRS-1-independent pathway (30, 50), nor the stimulation of DNA synthesis by EGF, a growth factor that signals mitogenesis through an Shc-dependent pathway (51). These latter findings further argue for IRS-1 dysfunction being responsible for the resistance of AIR-treated cells to insulin and IGF-I mitogenic signaling.
When comparing the state of insulin resistance achieved by chronic treatment with either insulin or AIRs, it appears that both insulin-resistant states correlated with IR down-regulation and defective tyrosine kinase activation (Refs. 2, 11, 52 and our results). It must be underlined that a mechanism by which insulin promotes cellular resistance to the hormone consisted of increased serine phosphorylation of IRS-1 (53) and IRs. However, chronic treatment of the cells with AIRs failed to alter IR and IRS-1 serine phosphorylation (data not shown), whereas it generated a misactivated IR that sequestered IRS proteins.
In conclusion, this study analyzed the molecular basis of the severe insulin resistance induced by AIRs in a newly diagnosed patient with type B syndrome. This analysis performed at the cellular level provides evidence for the ability of AIRs to induce a dual resistance to insulin and IGF-I through a novel mechanism involving the formation of stable and inactive complexes between IRs and IRSs, which act as negative signals for insulin and IGF-I actions.
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Acknowledgments |
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Footnotes |
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Received March 11, 1999.
Revised May 20, 1999.
Accepted May 25, 1999.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunit of the
human insulin receptor causes constitutive activation of the receptor
and insulin desensitization. J Clin Endocrinol Metab. 81:719–727.[Abstract]
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