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Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892; and Department of Oncology and Neurosciences, University G. D’Annunzio (D.L.E., A.C.), Chieti 66013, Italy
Address all correspondence and requests for reprints to: Michael J. Quon, M.D., Ph.D., Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10, Room 8C-218, 10 Center Drive, MSC 1755, Bethesda, Maryland 20892-1755. E-mail: quonm{at}nih.gov
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A previous study using phosphopeptides suggested that tyrosine-phosphorylated YXXM motifs at positions 608 and 939 in rat IRS-1 bind with high affinity to SH2 domains of p85, and motifs at positions 460 and 987 bind with lower affinity (10). Another study using intact cells concluded that the YXXM motif at position 608 accounts for 60% of the insulin-stimulated PI3K activity associated with IRS-1, whereas motifs at positions 939 and 987 do not contribute significantly (11). Overexpression of mutant rat IRS-1 with substitution of Phe for Tyr in YXXM motifs at positions 460, 608, 939, and 987 completely inhibited insulin-stimulated PI3K activity in Xenopus oocytes, but caused only a partial blockade in hepatocytes (12). Substitution of Phe for Tyr in 18 potential tyrosine phosphorylation sites in rat IRS-1 results in an IRS-1 mutant that does not undergo detectable tyrosine phosphorylation and is unable to mediate activation of PI3K in response to insulin (13). Adding back tyrosines to this F18 mutant at positions 608, 628, and 658 fully restores its ability to bind and activate PI3K in response to insulin at a level comparable to that seen with wild-type IRS-1 (13). Thus, previous studies are in agreement that the YXXM motif at position 608 in rat IRS-1 plays an important role in coupling insulin receptor signaling to PI3K activation. However, a single pair of YXXM motifs in IRS-1 that can simultaneously engage the tandem SH2 domains of p85 and fully activate PI3K at a level comparable to that of wild-type IRS-1 has not been identified. In the present study we found that the pair of YXXM motifs at positions 612 and 632 in human IRS-1 (corresponding to positions 608 and 628 in rat IRS-1) is sufficient to mimic the ability of wild-type IRS-1 to bind and activate PI3K and to mediate the translocation of GLUT4 in rat adipose cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IRS1-WT. IRS1-wild-type (WT), the complementary DNA for human IRS-1, was subcloned into pCIS2 as previously described (3), and the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to create sequence coding for an hemagglutinin (HA)-epitope tag fused to the C-terminus of IRS-1 (sense primer, 5'-G CAG CCA GAG GAC CGT CAG TAT CCT TAT GAT GTT CCT GAT TAT GCT TAG CTC AAC TGG ACA TCA CAG C-3'; antisense primer: 5'-G CTG TGA TGT CCA GTT GAG CTA AGC ATA ATC AGG AAC ATC ATA AGG ATA CTG ACG CTC CTC TGG CTG C-3').
IRS1-Y612/Y989. The QuickChange kit was used to sequentially introduce Phe for Tyr substitutions in the YXXM motifs at positions 465, 632, 662, and 941 in IRS1-WT with the following mutagenic oligonucleotides: 465 sense, 5'-G CTA AGC AAC TTT ATC TGC ATG GGT GGC-3'; 465 antisense, 5'-GCC ACC CAT GCA GAT AAA GTT GCT TAG C-3'; 632 sense, 5'-A AAG GGC AGT GGA GAC TTT ATG CCC ATG AG-3'; 632 antisense, 5'-CT CAT GGG CAT AAA GTC TCC ACT GCC CTT T-3'; 662 sense, 5'-TG GAC CCC AAT GGC TTC ATG ATG ATG TCC-3'; 662 antisense, 5'-GGA CAT CAT CAT GAA GCC ATT GGG GTC CA-3'; 941 sense, 5'-GGC ACT GAG GAG TTC ATG AAG ATG GAC C-3'; and 941 antisense, 5'-G GTC CAT CTT CAT GAA CTC CTC AGT GCC-3'.
IRS1-Y612. IRS1-Y612 was derived from IRS1-Y612/Y989 by introducing a Phe for Tyr substitution in the YXXM motif at position 989 with the mutagenic oligonucleotides: 989 sense, 5'-AGC CGG GGT GAC TTC ATG ACC ATG CAG-3'; and 989 antisense, 5'-CTG CAT GGT C AT GAA GTC ACC CCG GCT-3'.
IRS1-F6. IRS1-F6 was derived from IRS1-Y612 by introducing a Phe for Tyr substitution in the YXXM motif at position 612 with the mutagenic oligonucleotides: 612 sense, 5'-G GAT GAT GGC TTC ATG CCC ATG TCC CC-3'; and 612 antisense, 5'-GG GGA CAT GGG CAT GAA GCC ATC ATCC-3'.
IRS1-Y989. IRS1-Y989 was derived from IRS1-Y612/Y989 by introducing a Phe for Tyr substitution in the YXXM motif at position 612 with the 612 sense and antisense mutagenic oligonucleotides described above.
IRS1-Y612/Y632. IRS1-Y612/Y632 was derived from IRS1-Y612 by adding back Tyr at position 632 using the Morph mutagenesis kit (5 Prime, 3 Prime, Inc., Boulder, CO) and the mutagenic oligonucleotide 5'-G GGC AG T GG A GAC TAT ATG CCC ATG AGC-3'.
IRS1-Y632. IRS1-Y632 was derived from IRS1-F6 by adding back Tyr at position 632 as described above for IRS1-Y612/Y632.
GLUT4-HA. GLUT4-HA is the complementary DNA for human GLUT4 with the HA epitope tag subcloned into pCIS2 (16).
Key regions of all constructs were directly sequenced to confirm the mutagenesis.
Cell culture and transfection of NIH-3T3IRcells
NIH-3T3 fibroblasts stably overexpressing human insulin
receptors (NIH-3T3IR) (17) were
cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100
µg/ml streptomycin, and 2 mM glutamine in a humidified
atmosphere with 5% CO2. Before transfection,
NIH-3T3IR cells were seeded in 100-mm dishes at
50% confluence and cultured in medium without antibiotics for 1 day.
Lipofectamine Plus reagent (Life Technologies, Inc.,
Gaithersburg, MD) was used to transiently transfect cells with 4 µg
plasmid DNA/dish as described in the manufacturer’s instructions. One
day after transfection, cells were serum-starved overnight before
insulin treatment.
Coimmunoprecipitation of IRS-1 constructs and p85
NIH-3T3IR cells transiently transfected
with various IRS-1 constructs were serum-starved overnight and treated
without or with insulin (100 nM, 3 min, 37 C). The cells
were then washed with ice-cold PBS, and lysates were prepared using 500
µl lysis buffer [20 mM Tris-HCl (pH 7.5), 137
mM NaCl, 1 mM MgCl2, 1
mM CaCl2, 10% glycerol, 0.1
mM Na3VO4, 2
mM phenylmethylsulfonylfluoride, and 1% Nonidet P-40].
Lysates were cleared by centrifugation (10,000 x g for
20 min), and total protein content was determined by the Bradford
method using the Bio-Rad Laboratories, Inc., protein assay
kit (Richmond, CA). HA-tagged IRS proteins were immunoprecipitated by
incubating 3 µg polyclonal anti-HA antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) with cell lysates (400 µg
total protein) at 4 C overnight. Fifty microliters of 50% (wt/vol)
protein A-Sepharose in 50 mM Tris-HCl, pH 7.4,
were added to each sample and incubated for an additional 4 h at 4
C. The immune complexes were washed three times with ice-cold
immunoprecipitation buffer [10 mM Tris-HCl (pH
7.5), 150 mM NaCl, 1 mM
EGTA, 0.2 mM
Na3VO4, 0.2
mM phenylmethylsulfonylfluoride, 1% Triton, and
0.5% Nonidet P-40]. Each sample was then boiled for 5 min in Laemmli
sample buffer, subjected to SDS-PAGE, and immunoblotted with antibodies
against IRS-1 (polyclonal antibody, Upstate Biotechnology, Inc., Lake Placid, NY) or the p85
subunit of PI3K
(Upstate Biotechnology, Inc.). Immunoblots were visualized
using the ECL system (Amersham Pharmacia Biotech,
Arlington Heights, IL) and were quantified by scanning
densitometry.
IRS-1 associated PI3K activity
NIH-3T3IR cells transiently transfected
with IRS-1 constructs were subjected to insulin stimulation and
immunoprecipitation with anti-HA antibody as described above. The
immune complexes were washed once with PBS containing 1% Nonidet P-40
and 100 µM Na3VO4, twice
with 100 mM Tris-HCl (pH 7.5), containing 500
mM LiCl2 and 100 mM
Na3VO4, and once with 10
mM Tris-HCl (pH 7.5) containing 100 mM NaCl, 1
mM EDTA, and 100 mM
Na3VO4. For each reaction,
10 µg phosphatidylinositol (PI) (Sigma) were sonicated
in 10 µl PI3K reaction buffer [20 mM Tris-HCl (pH 7.5),
100 mM NaCl, and 0.3 mM EGTA], and 10 µCi
[
-32P]ATP in 40 µl PI3K reaction buffer
were added along with MgCl2 at a final
concentration of 10 mM. The phosphorylation reaction was
started by adding 50 µl of the substrate solution with 50 µl of the
immune complex. After incubation for 20 min at 30 C, the reaction was
stopped by adding 100 µl 0.1 N HCl and 200 µl
CHCl3/methanol (1:1). The organic phase
containing PI3-P was extracted and applied to a silica gel TLC plate
(Whatman, Clifton, NJ) coated with 1% potassium oxalate.
TLC plates were developed in
CHCl3/CH3OH/H2O/NH4OH
(60:47:11.3:2), dried, and visualized by autoradiography, quantified by
scanning densitometry, and normalized for the amount of IRS-1 recovered
in the anti-HA immunoprecipitates.
Electroporation of isolated rat adipose cells
Isolated adipose cells were prepared from epididymal fat pads of
male rats (170–200 g, CD strain, Charles River Laboratories, Inc., Wilmington, MA) by collagenase digestion and transiently
transfected by electroporation with GLUT4-HA and IRS constructs as
previously described (15, 18). Particulate fractions
derived from transfected cells were isolated and subjected to
immunoblotting with anti-HA or anti-IRS-1 antibodies as previously
described (4, 5).
Assay for cell surface epitope-tagged GLUT4
Twenty hours after electroporation, adipose cells were processed
as previously described (16) and treated with insulin at
final concentrations of 0, 0.024, 0.072, 0.3, and 60 nM at
37 C for 30 min. Cell surface epitope-tagged GLUT4 was determined using
monoclonal anti-HA antibody (HA-11, BAbCo, Berkeley, CA) in conjunction
with 125I-labeled sheep antimouse IgG as
previously described (19).
Statistical analysis
Paired t tests were used to compare individual points
where appropriate. Multiple ANOVA was used to compare insulin
dose-response experiments. P < 0.05 was considered to
indicate statistical significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) to determine the effects of specific
tyrosine residues on the ability of IRS-1 to engage and activate PI3K
in response to insulin. As determined by immunoblotting, comparable
levels of recombinant IRS-1 were recovered in anti-HA
immunoprecipitates (data not shown). In control cells, transfected with
the empty expression vector pCIS2, anti-p85 immunoblotting of anti-HA
immunoprecipitates revealed a weak nonspecific signal in both the
absence and presence of insulin (Fig. 2A, lanes 1 and 2). As expected, in cells transfected with IRS1-WT, the
small amount of p85 associated with IRS-1 in the absence of insulin was
significantly increased approximately 8-fold upon insulin stimulation
(Fig. 2A, lanes 3, 4, 9, and 10). By contrast, similar to the control
cells, little detectable association of p85 with IRS1-F6 (Phe
substituted for Tyr465,
Tyr612, Tyr632,
Tyr662, Tyr989, and
Tyr941 in YXXM motifs of IRS-1) was observed in
either the absence or presence of insulin (Fig. 2A, lanes 5, 6, 11, and
12). These results suggest that one or more of these six Tyr residues
is essential for p85 binding to IRS-1 in response to insulin.
Interestingly, association of p85 with either IRS1-Y612 or IRS1-Y632 in
response to insulin stimulation was approximately 40% and 30%,
respectively, of that observed with IRS1-WT (Fig. 2A, lanes 7, 8, 13,
and 14), whereas p85 association with IRS1-Y612/Y632 in response to
insulin was similar to that observed with IRS1-WT (Fig. 2A, lanes 15
and 16). The presence or absence of Tyr989 did
not significantly alter these results (data not shown). We next
assessed PI3K activity in the anti-HA immunoprecipitates. Little, if
any, PI3K activity was detected in immunoprecipitates derived from
either control cells (transfected with pCIS2) or cells transfected with
IRS1-F6 (Fig. 2B). By contrast, insulin stimulation caused an
approximately 5-fold increase in PI3K activity associated with either
IRS1-WT or IRS1-Y612/Y632. Compared with IRS1-WT, the PI3K activities
associated with IRS1-Y612 or IRS1-Y632 in response to insulin
stimulation were decreased by 44% and 75%, respectively. Thus, as
expected, the relative levels of PI3K activity associated with IRS-1 in
response to insulin generally paralleled the ability of p85 to
coimmunoprecipitate with the various IRS-1 constructs. Taken together,
our data suggest that the presence of Tyr612 and
Tyr632 together is sufficient for IRS-1 to bind
and activate PI3K at levels comparable to wild-type IRS-1.
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and 4, 
). In each independent
dose-response experiment a paired internal control group was always
included to compare with the experimental group. As previously reported
(3, 4, 5), overexpression of wild-type IRS-1 in the absence
of insulin was sufficient to increase cell surface GLUT4-HA to levels
about 75% of those seen in control cells treated with a maximally
stimulating dose of insulin (Fig. 3A). Insulin stimulation further
increased cell surface GLUT4 to levels about 140% of those observed in
the insulin-stimulated control cells. By contrast, overexpression of
IRS1-F6 or IRS1-Y989 resulted in insulin dose-response curves similar
to those obtained with the paired controls (Fig. 3, B and C). Thus, one
or more Tyr residues at positions 465, 612, 632, 662, or 941 may be
important for mediating metabolic actions of IRS-1, whereas
Tyr989 does not appear to be essential for these
functions.
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). Because of
limitations on the number of cuvettes that can be processed in a single
experiment, we included a paired control group transfected with the
empty vector pCIS2 for each experiment, but could not also include a
group for overexpression of wild-type IRS-1. In the absence of insulin,
basal levels of GLUT4 on the surface of cells expressing either
IRS1-Y612 or IRS1-Y632 were about 35% greater than basal levels in the
control cells (Fig. 4, A and B). However, these elevated basal levels
were still significantly lower than those observed in cells
overexpressing wild-type IRS-1 (cf. Fig. 3A). At low insulin
concentrations, the levels of GLUT4 recruited to the plasma membrane in
cells expressing either IRS1-Y612 or IRS1-Y632 were also significantly
greater than those in control cells. However, at the
60-nM insulin dose, cell surface GLUT4 levels
were comparable to those in control cells. Results from cells
expressing IRS1-Y612/Y989 were similar to those obtained with IRS1-Y612
(data not shown). Thus, adding back either Tyr612
or Tyr632 to the IRS1-F6 mutant partially
restored the ability of IRS-1 to mediate translocation of GLUT4.
Interestingly, in cells expressing IRS1-Y612/Y632, the basal level of
GLUT4 at the cell surface in the absence of insulin was greater than
that observed with the expression of either IRS1-Y612 or IRS1-Y632
(Fig. 4C). Indeed, the insulin dose-response curve obtained from cells
expressing IRS1-Y612/Y632 was similar to that of cells overexpressing
wild-type IRS-1 (cf. Fig. 3A). Thus, the presence of both
Tyr612 and Tyr632 is
sufficient to mimic the ability of wild-type IRS-1 to mediate
translocation of GLUT4. Moreover, the ability of the various IRS-1
mutants to mediate translocation of GLUT4 in rat adipose cells
correlated with data obtained from transfected
NIH-3T3IR cells regarding p85
coimmunoprecipitation and IRS-1-associated PI3K activity. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mutational analysis of the platelet-derived growth factor receptor was
previously employed by others to identify specific phosphotyrosine
motifs that interact with SH2 domains of a variety of signaling
molecules, including PI3K (27). In the present study we
evaluated the importance of various YXXM motifs in IRS-1 to binding,
activation and function of PI3K. Previous studies identified six YXXM
motifs in rat IRS-1 (equivalent to positions 465, 612, 632, 662, 941,
and 989 in human IRS-1) as potential sites for interaction with PI3K
(10, 11, 12, 13). These studies are in agreement that
Tyr608 in rat IRS-1 (equivalent to
Tyr612 in human IRS-1) plays an important role.
As our IRS1-F6 mutant did not significantly engage or activate PI3K,
our data also suggest that one or more of the six tyrosines at 465,
612, 632, 662, 941, and 989 are essential for coupling insulin
signaling to activation of PI3K by IRS-1. We ruled out a critical role
for Tyr989 because its presence or absence did
not alter PI3K binding and activity in response to insulin stimulation.
Adding back either Tyr612 or
Tyr632 alone to the F6 mutant partially restored
its capacity to engage PI3K. Importantly, adding back both
Tyr612 and Tyr632 was
sufficient to fully mimic the ability of wild-type IRS-1 to bind and
activate PI3K. Thus, we identified a pair of YXXM motifs at
Tyr612 and Tyr632 that may
be capable of simultaneously engaging the tandem SH2 domains of p85. To
exhaustively test all possible combinations of the six YXXM motifs
targeted in our studies it would be necessary to construct and examine
6! mutants (720 mutants). This is clearly beyond the scope of
the present study. However, based upon the published literature, it
seems unlikely that YXXM motifs at 465, 662, and 941 contribute
significantly to binding and activation of PI3K in intact cells. Rocchi
et al. ruled out important roles for
Tyr939 and Tyr987 in rat
IRS-1 (equivalent to Tyr941 and
Tyr989 in human IRS-1) (11). The
fact that no PI3K binding or activity was associated with a mutant rat
IRS-1 with disrupted YXXM motifs at 460, 608, 939, and 987 (equivalent
to 465, 612, 941, and 989 in human IRS-1) suggests that the YXXM motif
at 662 is not critical (12). Finally, the YXXM motif at
460 in rat IRS-1 (equivalent to 465 in human IRS-1) has very low
affinity for the SH2 domains of p85 in vitro relative to the
YXXM motif at 608 (10). Nevertheless, we cannot completely
rule out the possibility that other pairs of YXXM motifs in IRS-1,
besides the pair we identified at 612 and 632, may also mimic the
ability of wild-type IRS-1 to bind and activate PI3K. We recently
demonstrated that serine phosphorylation of IRS-1 by protein kinase
C-
impairs the ability of IRS-1 to activate PI3K
(28). Interestingly, similar impairment was observed with
the Y612/Y632 addback mutant (28). These results suggest
that Tyr612 and Tyr632 are
not only important for IRS-1 to fully activate PI3K, but that these two
phosphorylation sites may also be subject to direct or indirect
mechanisms for negative feedback regulation by downstream inputs.
The effects of overexpression of our IRS-1 constructs on GLUT4 translocation in rat adipose cells were also consistent with our findings regarding coimmunoprecipitation of p85 and PI3K activity with IRS-1. That is, overexpression of the F6 mutant or the Y989 addback mutant did not alter insulin-stimulated translocation of GLUT4, whereas overexpression of the Y612/Y632 addback mutant mimicked the effect of wild-type IRS-1 to recruit GLUT4 in the absence of insulin. Mutants with Y612 or Y632 added back alone had intermediate effects. Thus, the pair of YXXM motifs at positions 612 and 632 is sufficient for the ability of IRS-1 to mediate the metabolic actions of insulin in a physiologically relevant insulin target cell. We previously demonstrated that an antisense ribozyme against IRS-1 partially inhibits GLUT4 translocation in rat adipose cells (3), whereas overexpression of IRS3-F4 (analogous to IRS1-F6) completely inhibits insulin-stimulated translocation of GLUT4 (5). We had postulated that IRS3-F4 was binding to the insulin receptor and preventing all endogenous IRS isoforms from coupling insulin receptor signaling to PI3K activation. However, in the present study, although IRS1-F6 was not able to effect recruitment of GLUT4 per se, it did not exhibit the inhibitory actions of IRS3-F4. One possible explanation for this is that IRS-2 or IRS-3 may compete for binding to the insulin receptor more effectively than IRS-1. Interestingly, Sharma et al. reported that overexpression of isolated PTB or SAIN domains of IRS-1 impairs IRS-1-associated PI3K activity without affecting insulin-stimulated glucose transport in 3T3-L1 adipocytes (29). Although the researchers concluded from these data that IRS-1 does not play an important role in mediating the metabolic actions of insulin, it is possible that their results could also be explained by the ability of IRS-2 or -3 to compete more effectively than IRS-1 for binding to the insulin receptor, resulting in subsequent activation of glucose transport. In brown adipose cells derived from IRS-2 knockout mice, the impaired insulin-stimulated glucose transport and translocation of GLUT4 can be ameliorated by overexpression of IRS-2, but not IRS-1 (26). Thus, different IRS isoforms may interact in distinct ways with the insulin receptor, and these isoforms may not have completely overlapping or redundant functions.
The identification of a pair of YXXM motifs at positions 612 and 632 in
human IRS-1 that is sufficient to mediate PI3K-dependent metabolic
actions of insulin (presumably by simultaneously engaging the tandem
SH2 domains of p85) may be useful for understanding the normal
mechanisms of insulin action as well as the pathophysiology resulting
from mutations of human IRS-1 in this region. For example, a
heterozygous M613V mutation that disrupts the YMXM motif at position
612 was discovered in a patient with severe type A insulin resistance
(30). A number of other naturally occurring silent
polymorphisms as well as amino acid substitutions have been identified
at various sites in human IRS-1 (30, 31, 32, 33, 34, 35, 36, 37). The most common
variant, G972R, is estimated to be present in approximately 10% of
subjects with type 2 diabetes and approximately 5% of the nondiabetic
population (38). Some studies document modest impairment
of the G972R IRS-1 mutant to bind and activate PI3K (39)
and mild defects in the ability of the mutant to mediate metabolic
actions of insulin (40). However, other studies are unable
to detect abnormal function of the G972R IRS-1 mutant
(41), and the presence of this mutation does not appear to
decrease insulin sensitivity in humans, as assessed by glucose clamp
(42, 43). Intriguingly, the ability of IRS-1 to activate
PI3K may also be important for normal ß-cell function
(44), and the G972R IRS-1 mutation has been implicated in
impaired ß-cell survival and insulin secretion (45, 46).
Although position 972 is somewhat distant from the YMXM motifs at
positions 612 and 632, it is conceivable that structural changes
resulting from the G972R mutation may affect the ability of PI3K to
fully engage the YMXM motifs at positions 612 and 632. For example,
serine phosphorylation of IRS-1 by Akt at sites distant from 612 and
632 affect the ability of IRS-1 to activate PI3K (47, 48).
Moreover, we recently demonstrated that serine phosphorylation of IRS-1
by protein kinase C- specifically inhibits the ability of
IRS-1 to activate PI3K through the YMXM motifs at 612 and 632
(28). It will be of great interest in future studies to
elucidate the mechanisms by which modifications or mutations in IRS-1
interact with the YMXM motifs at 612 and 632 to regulate the metabolic
functions of IRS-1.
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Acknowledgments |
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Footnotes |
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Received March 6, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ile) overexpressed
in transfected rat adipose cells fail to mediate translocation of
epitope-tagged GLUT4. Proc Natl Acad Sci USA 91:5587–5591 receptor is required for binding of Grb2 and SHP-2 but not for
activation of Ras or cell proliferation. Mol Cell Biol 16:6926–6936[Abstract]
phosphorylates IRS-1 and impairs its ability to activate PI
3-kinase in response to insulin. J Biol Chem 276:3543–3549Arg972 amino acid polymorphism in insulin
receptor substrate- 1 affects glucose metabolism in skeletal muscle
cells. J Clin Endocrinol Metab 85:2004–2013Arg amino acid polymorphism in IRS-1
impairs insulin secretion in pancreatic ß cells. J Clin Invest 104:357–364[Medline]
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M. T. Gentile, C. Vecchione, G. Marino, A. Aretini, A. Di Pardo, G. Antenucci, A. Maffei, G. Cifelli, L. Iorio, A. Landolfi, et al. Resistin Impairs Insulin-Evoked Vasodilation Diabetes, March 1, 2008; 57(3): 577 - 583. [Abstract] [Full Text] [PDF]
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J. Knobloch, I. Schmitz, K. Gotz, K. Schulze-Osthoff, and U. Ruther Thalidomide Induces Limb Anomalies by PTEN Stabilization, Akt Suppression, and Stimulation of Caspase-Dependent Cell Death Mol. Cell. Biol., January 15, 2008; 28(2): 529 - 538. [Abstract] [Full Text] [PDF]
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B. Xue, Y.-B. Kim, A. Lee, E. Toschi, S. Bonner-Weir, C. R. Kahn, B. G. Neel, and B. B. Kahn Protein-tyrosine Phosphatase 1B Deficiency Reduces Insulin Resistance and the Diabetic Phenotype in Mice with Polygenic Insulin Resistance J. Biol. Chem., August 17, 2007; 282(33): 23829 - 23840. [Abstract] [Full Text] [PDF]
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G. Shahid and T. Hussain GRK2 Negatively Regulates Glycogen Synthesis in Mouse Liver FL83B Cells J. Biol. Chem., July 13, 2007; 282(28): 20612 - 20620. [Abstract] [Full Text] [PDF]
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 J. L. Holzman, L. Liu, B. J. Duke, A. E. Kemendy, and D. C. Eaton Transactivation of the IGF-1R by aldosterone Am J Physiol Renal Physiol, April 1, 2007; 292(4): F1219 - F1228. [Abstract] [Full Text] [PDF]
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F. Andreozzi, E. Laratta, C. Procopio, M. L. Hribal, A. Sciacqua, M. Perticone, C. Miele, F. Perticone, and G. Sesti Interleukin-6 Impairs the Insulin Signaling Pathway, Promoting Production of Nitric Oxide in Human Umbilical Vein Endothelial Cells Mol. Cell. Biol., March 15, 2007; 27(6): 2372 - 2383. [Abstract] [Full Text] [PDF]
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A Corbould Chronic testosterone treatment induces selective insulin resistance in subcutaneous adipocytes of women J. Endocrinol., March 1, 2007; 192(3): 585 - 594. [Abstract] [Full Text] [PDF]
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 I. R Hutcheson, J. M Knowlden, H. E Jones, R. S Burmi, R. A McClelland, D. Barrow, J. M W Gee, and R. I Nicholson Inductive mechanisms limiting response to anti-epidermal growth factor receptor therapy Endocr. Relat. Cancer, December 1, 2006; 13(Supplement_1): S89 - S97. [Abstract] [Full Text] [PDF]
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 J. F. Kuemmerle Occupation of {alpha}vbeta3-integrin by endogenous ligands modulates IGF-I receptor activation and proliferation of human intestinal smooth muscle Am J Physiol Gastrointest Liver Physiol, June 1, 2006; 290(6): G1194 - G1202. [Abstract] [Full Text] [PDF]
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A. Tzatsos and K. V. Kandror Nutrients Suppress Phosphatidylinositol 3-Kinase/Akt Signaling via Raptor-Dependent mTOR-Mediated Insulin Receptor Substrate 1 Phosphorylation Mol. Cell. Biol., January 1, 2006; 26(1): 63 - 76. [Abstract] [Full Text] [PDF]
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G. Frangioudakis, J.-M. Ye, and G. J. Cooney Both Saturated and n-6 Polyunsaturated Fat Diets Reduce Phosphorylation of Insulin Receptor Substrate-1 and Protein Kinase B in Muscle during the Initial Stages of in Vivo Insulin Stimulation Endocrinology, December 1, 2005; 146(12): 5596 - 5603. [Abstract] [Full Text] [PDF]
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 T. Asano, Y. Yao, S. Shin, J. McCubrey, J. L. Abbruzzese, and S. A.G. Reddy Insulin Receptor Substrate Is a Mediator of Phosphoinositide 3-Kinase Activation in Quiescent Pancreatic Cancer Cells Cancer Res., October 15, 2005; 65(20): 9164 - 9168. [Abstract] [Full Text] [PDF]
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M. Luo, S. Reyna, L. Wang, Z. Yi, C. Carroll, L. Q. Dong, P. Langlais, S. T. Weintraub, and L. J. Mandarino Identification of Insulin Receptor Substrate 1 Serine/Threonine Phosphorylation Sites Using Mass Spectrometry Analysis: Regulatory Role of Serine 1223 Endocrinology, October 1, 2005; 146(10): 4410 - 4416. [Abstract] [Full Text] [PDF]
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 N. E. Avissar, L. Toia, and H. C. Sax Epidermal Growth Factor and/or Growth Hormone Induce Differential, Side-Specific Signal Transduction Protein Phosphorylation in Enterocytes JPEN J Parenter Enteral Nutr, September 1, 2005; 29(5): 322 - 336. [Abstract] [Full Text] [PDF]
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 J. Luo, S. J. Field, J. Y. Lee, J. A. Engelman, and L. C. Cantley The p85 regulatory subunit of phosphoinositide 3-kinase down-regulates IRS-1 signaling via the formation of a sequestration complex J. Cell Biol., August 1, 2005; 170(3): 455 - 464. [Abstract] [Full Text] [PDF]
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 R. A. Velliquette, J. E. Friedman, J. Shao, B. B. Zhang, and P. Ernsberger Therapeutic Actions of an Insulin Receptor Activator and a Novel Peroxisome Proliferator-Activated Receptor {gamma} Agonist in the Spontaneously Hypertensive Obese Rat Model of Metabolic Syndrome X J. Pharmacol. Exp. Ther., July 1, 2005; 314(1): 422 - 430. [Abstract] [Full Text] [PDF]
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L. Khamzina, A. Veilleux, S. Bergeron, and A. Marette Increased Activation of the Mammalian Target of Rapamycin Pathway in Liver and Skeletal Muscle of Obese Rats: Possible Involvement in Obesity-Linked Insulin Resistance Endocrinology, March 1, 2005; 146(3): 1473 - 1481. [Abstract] [Full Text] [PDF]
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 J. L. Smith, J.-S. Ju, B. M. Saha, B. A. Racette, and J. S. Fisher Levodopa with carbidopa diminishes glycogen concentration, glycogen synthase activity, and insulin-stimulated glucose transport in rat skeletal muscle J Appl Physiol, December 1, 2004; 97(6): 2339 - 2346. [Abstract] [Full Text] [PDF]
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J. Lei, C. N. Mariash, and D. H. Ingbar 3,3',5-Triiodo-L-thyronine Up-regulation of Na,K-ATPase Activity and Cell Surface Expression in Alveolar Epithelial Cells Is Src Kinase- and Phosphoinositide 3-Kinase-dependent J. Biol. Chem., November 12, 2004; 279(46): 47589 - 47600. [Abstract] [Full Text] [PDF]
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F. Andreozzi, C. D'Alessandris, M. Federici, E. Laratta, S. Del Guerra, S. Del Prato, P. Marchetti, R. Lauro, F. Perticone, and G. Sesti Activation of the Hexosamine Pathway Leads to Phosphorylation of Insulin Receptor Substrate-1 on Ser307 and Ser612 and Impairs the Phosphatidylinositol 3-Kinase/Akt/Mammalian Target of Rapamycin Insulin Biosynthetic Pathway in RIN Pancreatic {beta}-Cells Endocrinology, June 1, 2004; 145(6): 2845 - 2857. [Abstract] [Full Text] [PDF]
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F. Andreozzi, E. Laratta, A. Sciacqua, F. Perticone, and G. Sesti Angiotensin II Impairs the Insulin Signaling Pathway Promoting Production of Nitric Oxide by Inducing Phosphorylation of Insulin Receptor Substrate-1 on Ser312 and Ser616 in Human Umbilical Vein Endothelial Cells Circ. Res., May 14, 2004; 94(9): 1211 - 1218. [Abstract] [Full Text] [PDF]
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Y. Li, S. Eitan, J. Wu, C. J. Evans, B. Kieffer, X. Sun, and R. D. Polakiewicz Morphine Induces Desensitization of Insulin Receptor Signaling Mol. Cell. Biol., September 1, 2003; 23(17): 6255 - 6266. [Abstract] [Full Text] [PDF]
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K. Bouzakri, M. Roques, P. Gual, S. Espinosa, F. Guebre-Egziabher, J.-P. Riou, M. Laville, Y. Le Marchand-Brustel, J.-F. Tanti, and H. Vidal Reduced Activation of Phosphatidylinositol-3 Kinase and Increased Serine 636 Phosphorylation of Insulin Receptor Substrate-1 in Primary Culture of Skeletal Muscle Cells From Patients With Type 2 Diabetes Diabetes, June 1, 2003; 52(6): 1319 - 1325. [Abstract] [Full Text] [PDF]
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D. L. Esposito, Y. Li, C. Vanni, S. Mammarella, S. Veschi, F. Della Loggia, R. Mariani-Costantini, P. Battista, M. J. Quon, and A. Cama A Novel T608R Missense Mutation in Insulin Receptor Substrate-1 Identified in a Subject with Type 2 Diabetes Impairs Metabolic Insulin Signaling J. Clin. Endocrinol. Metab., April 1, 2003; 88(4): 1468 - 1475. [Abstract] [Full Text] [PDF]
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H. Kuriyama, I. Shimomura, K. Kishida, H. Kondo, N. Furuyama, H. Nishizawa, N. Maeda, M. Matsuda, H. Nagaretani, S. Kihara, et al. Coordinated Regulation of Fat-Specific and Liver-Specific Glycerol Channels, Aquaporin Adipose and Aquaporin 9 Diabetes, October 1, 2002; 51(10): 2915 - 2921. [Abstract] [Full Text] [PDF]
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M. Montagnani, L. V. Ravichandran, H. Chen, D. L. Esposito, and M. J. Quon Insulin Receptor Substrate-1 and Phosphoinositide-Dependent Kinase-1 Are Required for Insulin-Stimulated Production of Nitric Oxide in Endothelial Cells Mol. Endocrinol., August 1, 2002; 16(8): 1931 - 1942. [Abstract] [Full Text] [PDF]
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M. Navarro, B. Valentinis, B. Belletti, G. Romano, K. Reiss, and R. Baserga Regulation of Id2 Gene Expression by the Type 1 IGF Receptor and the Insulin Receptor Substrate-1 Endocrinology, December 1, 2001; 142(12): 5149 - 5157. [Abstract] [Full Text] [PDF]
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