0013-7227/03/$15.00/0
Printed in U.S.A.
The Journal of Clinical Endocrinology & Metabolism 88(4):1468 –1475
Copyright © 2003 by The Endocrine Society
doi: 10.1210/jc.2002-020933
CLINICAL CASE SEMINAR
A Novel T608R Missense Mutation in Insulin Receptor
Substrate-1 Identified in a Subject with Type 2 Diabetes
Impairs Metabolic Insulin Signaling
DIANA L. ESPOSITO, YUNHUA LI, CINZIA VANNI, SANDRA MAMMARELLA, SERENA VESCHI,
FULVIO DELLA LOGGIA, RENATO MARIANI-COSTANTINI, PASQUALE BATTISTA,
MICHAEL J. QUON, AND ALESSANDRO CAMA
Department of Oncology and Neurosciences, Section of Molecular Pathology, University Gabriele D’Annunzio, 66013 Chieti,
Italy; and Diabetes Unit, Laboratory of Clinical Investigation, National Center for Complementary and Alternative
Medicine, National Institutes of Health (Y.L., M.J.Q.), Bethesda, Maryland 20892
Naturally occurring mutations in insulin receptor substrate-1
(IRS-1) have previously been implicated in impaired insulin
action. We now report a novel mutation in IRS-1 with substitution of Arg for Thr608 that was identified in a patient with
type 2 diabetes mellitus. We detected the T608R mutation in
1 of 136 chromosomes from diabetic patients and in 0 of 120
chromosomes from nondiabetic controls, suggesting that this
is a rare IRS-1 variant. Conservation of Thr608 in human, monkey, rat, mouse, and chicken IRS-1 sequences is consistent
with a crucial function for this residue. Moreover, Thr608 is
located near the YMXM motif containing Tyr612 that is important for binding and activation of phosphoinositol 3-kinase
(PI 3-kinase). To investigate whether the T608R mutation impairs insulin signaling, we transiently transfected NIH-3T3IR
cells with hemagglutinin-tagged wild-type or T608R mutant
IRS-1 constructs. Recombinant IRS-1 immunoprecipitated
from transfected cells treated with or without insulin was
subjected to immunoblotting for the p85 regulatory subunit of
T
YPE 2 DIABETES mellitus usually results from a combination of defects in insulin action and insulin secretion (1). Both genetic and environmental factors influence the
risk of developing diabetes (1). The importance of genetic
factors in the pathogenesis of type 2 diabetes is highlighted
by the clustering of diabetes in families (2), the different
prevalence for diabetes among various racial groups (3, 4),
and the higher concordance rate for type 2 diabetes among
monozygotic twins compared with dizygotic twins (5, 6). In
addition, naturally occurring mutations in the insulin receptor gene have been identified that cause severe insulin resistance and diabetes (7). Moreover, in transgenic mouse
models, compound heterozygotes with one null allele of both
the insulin receptor and insulin receptor substrate-1 (IRS-1)
have a diabetic phenotype (8). Thus, IRS-1, a major substrate
for the insulin receptor tyrosine kinase, is a candidate gene
for inherited defects that predispose to diabetes. Indeed, a
Abbreviations: HA, Hemagglutinin; IRS-1, insulin receptor substrate-1; PI 3-kinase, phosphoinositol 3-kinase; SSCP, single-strand conformational polymorphism; TLC, thin layer chromatography; WT, wild
type.
PI 3-kinase as well as a PI 3-kinase assay. As expected, in
control cells transfected with wild-type IRS-1, insulin stimulation caused an increase in p85 coimmunoprecipitated with
IRS-1 as well as a 10-fold increase in IRS-1-associated PI 3kinase activity. Interestingly, when cells transfected with
IRS1-T608R were stimulated with insulin, both the amount of
p85 coimmunoprecipitated with IRS1-T608R as well as the
associated PI 3-kinase activity were approximately 50% less
than those observed with wild-type IRS-1. Moreover, in rat
adipose cells, overexpression of IRS1-T608R resulted in significantly less translocation of GLUT4 to the cell surface than
comparable overexpression of wild-type IRS-1. We conclude
that a naturally occurring substitution of Arg for Thr608 in
IRS-1 is a rare human mutation that may contribute to insulin
resistance by impairing metabolic signaling through PI 3kinase-dependent pathways. (J Clin Endocrinol Metab 88:
1468 –1475, 2003)
number of naturally occurring amino acid substitutions as
well as silent polymorphisms have been identified in IRS-1
from both diabetic and nondiabetic human subjects (9 –18).
However, some of these are either rare IRS-1 variants (14 –18)
or low frequency polymorphisms that show similar prevalence in diabetic and control subjects (9 –11, 19). Only a few
studies have investigated whether these naturally occurring
IRS-1 mutants impair insulin action (15, 18, 20). One common
mutation in IRS-1, the G972R variant, has been implicated in
both abnormal metabolic actions of insulin (15, 20 –22) as well
as defective insulin secretion (23, 24).
In the present study we identified a novel IRS-1 mutation
in a patient with type 2 diabetes that results in the substitution of arginine for threonine at codon 608 (T608R). In a
previous study we demonstrated that the pair of YXXM
motifs containing Tyr612 and Tyr632 in human IRS-1 is sufficient to mimic the ability of wild-type IRS-1 (IRS-WT) to
bind and activate phosphoinositol 3-kinase (PI 3-kinase) and
to mediate the translocation of GLUT4 in rat adipose cells
(25). As Thr608 is in close proximity to Tyr612, we hypothesized that the T608R mutation in IRS-1 may impair metabolic
insulin signaling and contribute to the development of dia-
1468
Esposito et al. • Clinical Case Seminar
betes. Interestingly, we found that the T608R IRS-1 mutant
had a decreased ability to bind and activate PI 3-kinase in
response to insulin stimulation and was also partially defective in mediating recruitment of the insulin-responsive
glucose transporter GLUT4. Thus, we have identified a novel
human mutation in IRS-1 that may directly contribute to
insulin resistance and predispose to type 2 diabetes.
Subjects and Methods
Human subjects and DNA analysis
A total of 111 Italian subjects (54 men and 57 women), including 64
patients with type 2 diabetes and 47 control subjects (fasting blood
glucose, ⬍110 mg/dl), were analyzed in this study. Written informed
consent was obtained from all participants, and the study was approved
by the ethical committee of the University of Chieti Gabriele
D’Annunzio. Genomic DNA was extracted from whole blood using the
QIAamp DNA blood kit (Qiagen, Hilden, Germany). PCR primers were
designed to amplify 13 overlapping DNA fragments spanning the entire
coding region of the human IRS-1 gene (primer sequences and PCR
conditions are available upon request). Labeling of the PCR products for
single-strand conformational polymorphism (SSCP) analysis was performed using a nested PCR protocol as previously described (26). PCR
products showing anomalous SSCP conformers were sequenced directly
on both strands using T7 Sequenase version 2.0 (Amersham Pharmacia
Biotech, Arlington Heights, IL). We adopted the nucleotide and amino
acid numbering according to the IRS-1 sequence derived from human
skeletal muscle reported by Araki et al. (27). For the T608R variant we
screened additional individuals up to a total of 68 patients and 60 control
subjects by SSCP and PCR-direct sequencing analysis.
Plasmid constructs
pCIS2. pCIS2 is a parental mammalian expression vector with cytomegalovirus promoter (28, 29).
IRS1-WT. cDNA for human IRS-1 was subcloned into pCIS2 as previously described (30), and a sequence coding for a hemagglutinin (HA)
epitope tag fused to the C terminus of IRS-1 was added as previously
described (25).
IRS1-T608R. An expression vector for mutant IRS-1 with substitution of
Arg for Thr608 was created using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and the following mutagenic
oligonucleotides: 608 sense, 5⬘-C TCC ACC CTC CAC AGG GAT GAT
GGC TAC ATG-3⬘; and 608 antisense, 5⬘-CAT GTA GCC ATC ATCCCT
GTG GAG GGT GGA G-3⬘. The mutagenesis was confirmed by direct
sequencing. Underline indicates mutation sites.
GLUT4-HA. cDNA for human GLUT4 with an HA epitope tag was
subcloned into pCIS2 (31).
Cell culture and transfection of NIH-3T3IR cells
NIH-3T3 fibroblasts stably overexpressing human insulin receptors
(NIH-3T3IR) (32) were cultured in DMEM supplemented with 10% fetal
bovine serum, 100 U/ml penicillin, 100 ␮g/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 d. Lipofectamine Plus
reagent (Life Technologies, Inc., Gaithersburg, MD) was then used to
transiently transfect cells with 4 ␮g plasmid DNA/dish as described by
the manufacturer.
Coimmunoprecipitation of p85 with recombinant IRS-1
IR
NIH-3T3 cells transiently transfected with pCIS2, IRS1-WT, or IRS1T608R were serum-starved overnight and then treated with or without
insulin (100 nm, 3 min, 37 C). The cells were then washed with ice-cold
PBS, and cell lysates were prepared using 500 ␮l 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
J Clin Endocrinol Metab, April 2003, 88(4):1468 –1475 1469
P-40]. Lysates were cleared by centrifugation (10,000 ⫻ g for 20 min), and
total protein content was determined by the Bradford method (Bio-Rad
Laboratories, Inc., Richmond, CA). HA-tagged IRS proteins were immunoprecipitated by incubating 3 ␮g polyclonal anti-HA antibody
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) with cell lysates (400
␮g total protein) at 4 C overnight. Protein A-Sepharose [50 ␮l of a 50%
(wt/vol) slurry in 50 mm Tris-HCl (pH 7.4)] was added to each sample,
and samples were incubated for an additional 4 h at 4 C. 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 using antibodies against IRS-1 (polyclonal antibody, Upstate Biotechnology, Inc.,
Lake Placid, NY) or the p85␣ subunit of PI 3-kinase (Upstate Biotechnology, Inc.). Immunoblots were visualized using the enhanced chemiluminescence system (Amersham Pharmacia Biotech) and quantified by
scanning densitometry. Data were normalized for the amount of IRS-1
recovered in the anti-HA immunoprecipitates.
IRS-1-associated PI 3-kinase 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 ␮m 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 ␮g PI (Sigma-Aldrich,
St. Louis, MO) were sonicated in 10 ␮l PI 3-kinase reaction buffer [20 mm
Tris-HCl (pH 7.5), 100 mm NaCl, and 0.3 mm EGTA] and 10 ␮Ci
[␥-32P]ATP in 40 ␮l PI 3-kinase reaction buffer (supplemented with 10
mm MgCl2) were added. The phosphorylation reaction was started by
adding 50 ␮l of the substrate solution to 50 ␮l of the immune complex.
After incubation for 20 min at 30 C, the reaction was stopped by adding
100 ␮l 0.1 n HCl and 200 ␮l CHCl3/methanol (1:1). The organic phase
containing labeled PI(3)P was extracted and applied to a silica gel thin
layer chromatography (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, visualized by autoradiography, and
quantified by scanning densitometry. Data were normalized for the
amount of IRS-1 recovered in the anti-HA immunoprecipitates.
Transfection of rat adipose cells and GLUT4
translocation assay
Isolated adipose cells were prepared from epididymal fat pads of
male rats (CD strain, Charles River Laboratories, Inc., Wilmington, MA)
by collagenase digestion and transiently transfected by electroporation
with GLUT4-HA and IRS-1 constructs as previously described (29, 33).
Twenty hours after electroporation, adipose cells were processed as
previously described (31) and treated with insulin at final concentrations
of 0, 0.072, or 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 (34). Particulate fractions derived from
transfected cells were isolated and subjected to immunoblotting with
anti-HA or anti-IRS-1 antibodies as previously described (35, 36).
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.
Results
Detection of IRS-1 variants
SSCP analysis of the IRS-1 gene in 64 patients with type 2
diabetes and 47 normoglycemic subjects revealed nucleotide
changes predicted to result in four previously described amino
acid substitutions (A512P, G818R, S892G, and G971R), four
1470
J Clin Endocrinol Metab, April 2003, 88(4):1468 –1475
Esposito et al. • Clinical Case Seminar
known silent polymorphisms (nucleotides C270T, G702A,
A2412G, and G2679C), and two novel IRS-1 variants. The
allele frequencies of IRS-1 variants detected in our study are
shown in Table 1. Among previously reported amino acid
variants (9 –18), A512P, G818R, and S892G were not frequent
in our population. The A512P and S892G variants were detected only in normoglycemic controls. Only one type 2 diabetes patient carried the G818R variant, whereas we detected the same variant in 2 normoglycemic controls. The
allelic frequency of the common IRS-1 variant G971R was
TABLE 1. Allele frequencies of the IRS-1 variants detected
Nucleotide
substitutionsa
270C 3 T
702G 3 A
1440C 3 T
1534G 3 C
1823C 3 G
2412A 3 G
2452G 3 C
2674A 3 G
2679G 3 C
2911G 3 A
a
Amino acid
substitutionsa
A512P
T608R
G818R
S892G
G971R
Allele frequency
Type 2 diabetesb
Control subjectsc
0.031
0.070
0.0078
0
0.0077
0.133
0.0078
0
0.031
0.070
0.032
0.085
0
0.01
0
0.128
0.021
0.021
0.042
0.085
Nucleotide and amino acid numbering is according to Araki et al.
(27).
b
Number of alleles screened, n ⫽ 128 (the screening for the T608R
variant was extended to n ⫽ 136 alleles).
c
Number of alleles screened, n ⫽ 94 (the screening for the T608R
variant was extended to n ⫽ 120 alleles).
similar in our patients and control subjects. Of the novel
variants, one was a silent nucleotide change (C1440T), and
the other was a mutation at codon 608 (C1823G) resulting in
the substitution of arginine for threonine (T608R; Fig. 1, A
and B). For the T608R variant we screened additional individuals up to a total of 68 patients and 60 control subjects by
SSCP and PCR-direct sequencing analysis. In our study population the T608R mutation was detected in only 1 of 136
chromosomes from patients with type 2 diabetes and in 0 of
120 chromosomes from control subjects. Interestingly, the
T608R mutation is located in a domain that is perfectly conserved across several species, and it is adjacent to an important PI 3-kinase binding site (Fig. 1C). The novel nonconservative amino acid substitution, T608R, was detected in a male
patient (patient 58D) diagnosed with type 2 diabetes at age
56 yr (body mass index, 26.8 kg/m2; plasma triglycerides,
1.58 mmol/liter; total cholesterol, 4.65 mmol/liter). The patient had retinopathy at the time of diagnosis. He subsequently developed diabetic foot complications as well as
coronary artery disease and died at age 71 yr after myocardial
infarction. The patient’s level of fasting serum C peptide (600
pmol/liter) were comparable to those of gender-, age-, and
body mass index-matched patients with diabetes (data not
shown). Considering that patient 58D was treated with a
combination of insulin (13 U Monotard/lente and 3 U Actrapid/regular/d; Novo Nordisk, Copenhagen, Denmark)
and oral agents (gliclazide) at the time of analysis, this level
of C peptide is consistent with the presence of insulin resis-
FIG. 1. The novel T608R amino acid substitution in IRS-1 is located in a highly conserved region. A,The IRS-1 region spanning codons 529 – 644
was amplified by PCR in 68 unrelated type 2 diabetes patients and 60 unrelated controls. Amplified DNA was analyzed by SSCP. The WT pattern
(lane 1) derived from a control subject is distinct from the unique SSCP conformer (lane 2, arrows) detected in patient 58D. B, Partial sequencing
ladders obtained by direct sequencing of the PCR amplified IRS-1 region containing the novel T608R amino acid substitution. The arrow indicates
the heterozygous 1823C to G nucleotide substitution generating the missense mutation. C, Aligned sequences from patient 58D, human, rat,
mouse, monkey, and chicken IRS-1. The R residue at codon 608 is indicated in bold and underlined. The YMPM motif containing Tyr612 is
indicated in bold italics.
Esposito et al. • Clinical Case Seminar
tance in this patient. The mother of patient 58D and 3 of his
5 siblings were affected with type 2 diabetes. Unfortunately,
we could not ascertain cosegregation of the T608R variant
with type 2 diabetes in other family members. The only son
of patient 58D was normoglycemic and not a carrier of the
variant, whereas other family members refused analysis.
Impaired association of p85 and PI 3-kinase activity with
IRS1-T608R
We first investigated whether the T608R mutation in IRS-1
may impair metabolic insulin signaling pathways by examining NIH-3T3IR cells transiently transfected with WT or
mutant HA-tagged IRS-1 constructs. When lysates from cells
treated without or with insulin were immunoblotted with
antiphosphotyrosine antibody, we observed comparable insulin receptor autophosphorylation in cells transfected with
either IRS1-T608R or IRS1-WT (Fig. 2, upper panel). Moreover,
when recombinant IRS-1 was immunoprecipitated from cell
lysates using an anti-HA antibody, we also observed comparable insulin-stimulated phosphorylation of IRS-T608R
and IRS1-WT (Fig. 2, lower panel). We next examined the
ability of the p85 regulatory subunit of PI 3-kinase to coimmunoprecipitate with the mutant IRS-1. As expected, when
samples were immunoblotted with anti-IRS-1 antibody,
comparable amounts of WT and mutant IRS-1 were detected
(Fig. 3A, bottom panel, and Fig. 3D; P ⬍ 0.46). Coimmunoprecipitation of p85 was determined by immunoblotting
samples with an anti-p85 antibody (Fig. 3A, top panel). 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. 3A, top panel, and Fig. 3B, lanes
1 and 2). For WT IRS-1, the amount of associated p85 after
insulin stimulation was significantly increased approximately 10-fold over the basal amount (Fig. 3A, top panel, and
J Clin Endocrinol Metab, April 2003, 88(4):1468 –1475 1471
Fig. 3B, lanes 3 and 4; P ⬍ 10⫺6). By contrast, the amount of
p85 associated with IRS1-T608R after insulin stimulation was
significantly decreased by approximately 50% (P ⬍ 0.0001)
compared with WT IRS-1 (Fig. 3A, top panel, and Fig. 3B,
compare lanes 5 and 6 with lanes 3 and 4). Thus, in response
to insulin stimulation, the T608R mutant of IRS-1 had an
impaired ability to bind to the p85 regulatory subunit of PI
3-kinase, a key metabolic signaling molecule.
To determine whether decreased binding of p85 with IRS1T608R also resulted in decreased PI 3-kinase activity, we next
assessed PI 3-kinase activity associated with WT and mutant
IRS-1 before and after insulin stimulation (Fig. 3A, middle
panel, and Fig. 3C). Importantly, the PI 3-kinase activity detected in anti-HA immunoprecipitates derived from cell lysates of transfected NIH-3T3IR cells was consistent with our
p85 co-immunoprecipitation findings. That is, minimal PI
3-kinase activity was detected in anti-HA immunoprecipitates derived from cells transfected with empty vector (Fig.
3A, middle panel, and Fig. 3C, lanes 1 and 2). For cells transfected with WT IRS-1, stimulation with insulin caused an
approximately 10-fold increase in IRS-1-associated PI 3-kinase activity (Fig. 3A, middle panel, and Fig. 3C, lanes 3 and
4; P ⬍ 10⫺5). By contrast, the PI 3-K activity associated with
IRS1-T608R in response to insulin stimulation was decreased
by about 50% compared with that of the WT IRS-1 (Fig. 3A,
middle panel, and Fig. 3C, lanes 5 and 6; P ⬍ 0.008). Thus,
IRS1-T608R also had a significant defect in its ability to activate PI 3-kinase in response to insulin stimulation.
In addition to exploring metabolic signaling pathways, we
examined the ability of IRS1-T608R to mediate mitogenic
signals by assessing coimmunoprecipitation of Grb-2 with
IRS1-WT and IRS1-T608R before and after insulin stimulation. Similar to results with p85, the amount of Grb-2 coimmunoprecipitated with WT IRS-1 significantly increased after insulin stimulation (Fig. 4A, lower panel, and Fig. 4B; P ⬍
0.005). However, in contrast to our results with p85 and PI
3-kinase activity, the amount of Grb-2 associated with IRS1T608R in the insulin-stimulated state was similar to that
observed for WT IRS-1. Moreover, as previously shown in
Fig. 2, the amount of tyrosine-phosphorylated recombinant
IRS-1 was comparable for WT and mutant IRS-1 (Fig. 4A,
upper panel, and Fig. 4B; P ⬍ 0.1). Taken together, these data
suggest that the T608R mutation in IRS-1 may result in a
selective impairment in metabolic insulin signaling without
affecting other mitogenic insulin signaling pathways.
IRS1-T608R has an impaired ability to mediate
translocation of GLUT4 in rat adipose cells
FIG. 2. Insulin-stimulated tyrosine phosphorylation of the insulin
receptor and IRS-1 is unaffected by the T608R IRS-1 mutation. NIH3T3IR cells were transfected with WT IRS-1 (lanes 1 and 2) or IRS1T608R (lanes 3 and 4). Transfected cells were serum-starved overnight and then incubated either without (⫺) or with (⫹) insulin (100
nM) at 37 C for 3 min. Whole cell lysates were immunoblotted with
antiphosphotyrosine antibody to detect autophosphorylated insulin
receptor (upper panel). Anti-HA immunoprecipitates were immunoblotted with antiphosphotyrosine antibody to detect tyrosine-phosphorylated recombinant IRS-1. Representative blots are shown from
experiments that were repeated independently three times.
To determine whether the impaired association of p85 and
PI 3-kinase activity with IRS1-T608R also affected the ability
of the mutant to mediate metabolic actions of insulin, we
assessed the effects of overexpression of WT and mutant
IRS-1 constructs on GLUT4 translocation in transfected rat
adipose cells. Cells were cotransfected with GLUT4-HA
along with empty vector (pCIS2), IRS1-WT, or IRS1-T608R,
and cell surface GLUT4-HA was assessed before and after
insulin stimulation. Comparable overexpression of GLUT4-HA
and IRS-1 constructs was confirmed by immunoblotting with
anti-HA antibodies (Fig. 5B). As expected, in control cells
1472
J Clin Endocrinol Metab, April 2003, 88(4):1468 –1475
Esposito et al. • Clinical Case Seminar
FIG. 3. IRS1-T608R has an impaired ability to associate with p85 and activate PI 3-kinase. NIH-3T3IR cells were transfected with empty vector
(pCIS2; lanes 1 and 2), WT IRS-1 (lanes 3 and 4), or IRS1-T608R (lanes 5 and 6). Transfected cells were serum-starved overnight and then
incubated either without (⫺) or with (⫹) insulin (100 nM) at 37 C for 3 min. Recombinant IRS-1 was immunoprecipitated from cell lysates with
anti-HA antibodies. A, Anti-HA immunoprecipitates were separated by 8% SDS-PAGE, transferred to a nitrocellulose filter, and immunoblotted
with either anti-IRS-1 antibody (bottom panel) or anti-p85 antibody (upper panel). To analyze IRS-1-associated PI3K activity, anti-HA
immunoprecipitates were washed, and the pellets were incubated with PI and [␥-32P]ATP. Reaction products were separated by TLC, and a
representative autoradiogram corresponding to 32P-labeled phosphatidylinositol phosphate (PIP) is shown (middle panel). B, Quantification of
p85␣ subunit coimmunoprecipitated with recombinant IRS-1. The histogram shows the results (mean ⫾ SE) obtained by scanning densitometry
of immunoblots from four independent experiments normalized for IRS-1 recovery. C, IRS-1-associated PI 3-kinase activity was quantified by
digitizing the signal from x-ray films and analyzing the data using NIH Image software. Results shown are the mean ⫾ SE of three independent
experiments normalized for IRS-1 recovery. D, Quantification of recombinant IRS-1 immunoprecipitated from cell lysates with anti-HA
antibodies. The histogram shows the results (mean ⫾ SE) obtained by scanning densitometry of immunoblots from four independent experiments.
transfected with pCIS2 and GLUT4-HA, insulin stimulation
resulted in a dose-dependent increase in GLUT4 at the cell
surface (Fig. 5A). As previously reported (25, 30), compared
with control cells, overexpression of WT IRS-1 significantly
increased the amount of GLUT4 at the cell surface both in the
absence of insulin and at intermediate insulin doses. The
maximal insulin response in cells overexpressing WT IRS-1
was similar to that in control cells. Importantly, although
overexpression of IRS1-T608R in the absence of insulin resulted in recruitment of GLUT4 to the surface greater than
that observed in control cells in the basal state, the magnitude
of this effect was approximately 50% less than that seen with
overexpression of WT IRS-1 (Fig. 5A; P ⬍ 0.005). In addition,
there was a slight, but statistically significant, impairment in
the maximal amount of GLUT4 recruited after insulin stimulation in cells overexpressing IRS1-T608R compared with
control cells (P ⬍ 0.04). These results suggest that the T608R
mutation in IRS-1 may contribute to insulin resistance by
interfering with insulin-stimulated translocation of GLUT4
and glucose uptake in insulin target tissues.
Discussion
In our examination of Italian diabetic and control subjects
we detected two novel IRS-1 variants as well as a number of
previously described mutations and silent polymorphisms.
In agreement with previous reports (10, 13, 19, 37– 44), none
of the previously identified variants (including the frequent
IRS-1 mutation G972R) appeared to associate more frequently with type 2 diabetes in our study. The novel T608R
amino acid substitution we report in the present study represents a nonconservative substitution detected in a patient
with type 2 diabetes. T608R is not a common IRS-1 polymorphism, as it has never been reported previously, and we
detected it in only 1 of 136 chromosomes from patients with
type 2 diabetes and in 0 of 120 chromosomes from control
subjects. Based upon the sequence conservation among species at position 608 (perfectly conserved in human skeletal
muscle, rat, mouse, monkey, and chicken IRS-1) (27, 45– 48),
the nonconservative nature of the amino acid substitution,
and the close proximity of position 608 to the PI 3-kinase
Esposito et al. • Clinical Case Seminar
FIG. 4. IRS1-T608R does not have an impaired ability to associate
with Grb-2 in response to insulin stimulation. NIH-3T3IR cells were
transfected with WT IRS-1 (lanes 1 and 2) or IRS1-T608R (lanes 3 and
4). Transfected cells were serum-starved overnight and then incubated either without (⫺) or with (⫹) insulin (100 nM) at 37 C for 3 min.
Recombinant IRS-1 was immunoprecipitated from cell lysates with
anti-HA antibodies. A, Anti-HA immunoprecipitates were separated
by 8% SDS-PAGE, transferred to a nitrocellulose filter, and immunoblotted with either antiphosphotyrosine antibody (top panel) or
anti-Grb-2 antibody (bottom panel). B, Quantification of Grb-2 coimmunoprecipitated with recombinant IRS-1. The histogram shows the
results (mean ⫾ SE) obtained by scanning densitometry of immunoblots from two independent experiments.
binding motif YMPM at positions 612– 615, we hypothesized
that the T608R mutation may contribute to functional impairment of metabolic actions of insulin and predispose to diabetes.
To our knowledge, T608 has not been identified as a potential
phosphorylation site for any known threonine kinase.
IRS-1 plays a crucial role in mediating metabolic actions of
insulin through the activation of PI 3-kinase-dependent pathways (49). After IRS-1 undergoes tyrosine phosphorylation
by the insulin receptor, the tandem SH2 domains of the p85
regulatory subunit of PI 3-kinase specifically bind to tyrosylphosphorylated YMXM motifs on IRS-1, resulting in activation of the catalytic p110 subunit of PI 3-kinase (50). Full
activation of PI 3-kinase requires simultaneous occupancy of
both SH2 domains of p85 (51). In a previous study we identified a pair of YMXM motifs at Y612 and Y632 whose presence is sufficient to mimic the ability of WT IRS-1 to fully
activate PI 3-kinase and mediate translocation of GLUT4 in
response to insulin (25). IRS-1 mutants with only a single
intact YMXM motif at either Y612 or Y632 alone have an
approximately 50% impairment in their ability to bind and
activate PI 3-kinase compared with WT IRS-1 or with IRS-1
mutants that have both Y612 and Y632 intact. In the present
study the approximately 50% impairment in the ability of
IRS1-T608R to bind and activate PI 3-kinase in response to
insulin is consistent with the hypothesis that the nonconservative T608R substitution interferes with the ability of the
YMXM motif at position 612 to bind the SH2 domain of p85.
Alternatively, it is also possible that the T608R mutation
J Clin Endocrinol Metab, April 2003, 88(4):1468 –1475 1473
FIG. 5. IRS1-T608R has an impaired ability to mediate translocation
of GLUT4 in rat adipose cells. Adipose cells were cotransfected with
GLUT4-HA (1 ␮g/cuvette) and pCIS2, WT IRS-1, or IRS1-T608R (4
␮g/cuvette) and treated with insulin for 25 min (0, 0.07, or 60 nM). A,
Data are expressed as the percentage of cell surface GLUT4-HA in the
control group treated with 60 nM insulin. Results shown are the
mean ⫾ SEM of nine independent experiments. The dose-response for
IRS1-T608R is significantly different than that for WT IRS-1 by multiple ANOVA (P ⬍ 0.016). In addition, in the absence of insulin the
amount of GLUT4-HA at the cell surface in cells overexpressing IRS1T608R was less than with overexpression of WT IRS-1 (P ⬍ 0.005).
There was a slight, but statistically significant, impairment in the
maximal amount of GLUT4 recruited after insulin stimulation in cells
overexpressing IRS1-T608R compared with control cells (P ⬍ 0.04). B,
Cotransfected cells express comparable levels of GLUT4-HA and HAtagged recombinant IRS-1. Total membrane fractions derived from
cells cotransfected with GLUT4-HA (1 ␮g/cuvette) and pCIS2, WT
IRS-1, or IRS1-T608R (4 ␮g/cuvette) were subjected to immunoblotting with anti-HA antibody HA-11. Cells transfected with pCIS2 alone
represent a negative control for immunoblotting, as these cells do
not express GLUT4-HA (lanes 1). Roughly comparable levels of
GLUT4-HA are seen in all groups of cells cotransfected with GLUT4HA. Representative blots are shown from an experiment that was
repeated independently three times.
interferes with the association between either IRS-1 and the
insulin receptor or IRS-1 and PI 3-kinase by causing some
conformational change without affecting tyrosine phosphorylation at position 612 or any other tyrosine phosphorylation
site on IRS-1. Indeed, we found that the total IRS-1 tyrosine
phosphorylation in response to insulin stimulation was comparable between WT and mutant IRS-1. Nevertheless, as
there are more than 20 potential tyrosine phosphorylation
sites on IRS-1, it may be difficult to detect even complete lack
of phosphorylation in a single site by examining total IRS-1
tyrosine phosphorylation. There was some specificity to the
1474 J Clin Endocrinol Metab, April 2003, 88(4):1468 –1475
observed impairment in IRS-1 function, as insulin-stimulated
PI 3-kinase binding and activity associated with IRS-1 were
decreased without affecting the ability of the T608R mutant
to bind with Grb-2 in response to insulin stimulation. Grb-2
is an important adaptor molecule that couples IRS-1 to Ras
and MAPK signaling pathways involved with mitogenic actions of insulin. Thus, our data suggest that the T608R IRS-1
mutation may result in selective impairment of metabolic signaling without affecting other mitogenic signaling pathways.
Interestingly, another IRS-1 mutation located five residues
downstream from the T608R variant was previously reported
in a patient with extreme insulin resistance (18). However, by
contrast with the T608R mutant, no defect was observed in PI
3-kinase association or activity, whereas the interaction with
Grb2 was modestly impaired with that mutant (18).
As might be predicted for an IRS-1 mutant that has defects
in its ability to bind and activate PI 3-kinase, IRS1-T608R also
had an impaired ability to mediate translocation of GLUT4
in adipose cells. Interestingly, the defect we observed in
IRS1-T608R with respect to translocation of GLUT4 in adipose cells was most prominent in the basal state in the absence of insulin, whereas the impairment in binding and
activation of PI 3-kinase was best appreciated in the insulinstimulated state in NIH-3T3IR cells. As overexpression of WT
IRS-1 in adipose cells has a major effect to increase basal cell
surface GLUT4, the impaired function of the mutant IRS-1 is
most apparent when GLUT4 translocation is examined in the
basal state relative to cells overexpressing WT IRS-1. It may
be difficult to assess impairment of the mutant IRS-1 in the
insulin-stimulated state with our adipose cell experiments
because there is not much effect of overexpressing WT IRS-1
in the maximally insulin-stimulated state. In transfected adipose cells in the basal state, there is a 50% impairment in the
ability of IRS1-T608R to cause translocation of GLUT4 relative to WT IRS-1. The magnitude of this impairment is comparable to the decrease in IRS1-T608R binding and activation
of PI 3-kinase relative to WT IRS-1 observed in the insulinstimulated state in our NIH-3T3IR cells. The ability of recombinant IRS-1 to bind and activate PI 3-kinase is easiest to
ascertain in the insulin-stimulated state. Differences in the
basal state in NIH-3T3IR cells are difficult to determine because the signal is so small in the absence of insulin stimulation. This difficulty would also be expected if we were to
measure p85 and PI 3-kinase activity associated with transfected IRS-1 constructs in adipose cells. As GLUT4 translocation is known to be dependent on PI 3-kinase activity, our
data from both adipose cells and NIH-3T3 cells support the
conclusion that the mutant IRS-1 has an impairment in the
ability to couple with PI 3-kinase. Accordingly, metabolic
insulin resistance present in patient 58D with the T608R
mutation may have been due in part to defective signaling
to PI 3-kinase and impaired translocation of GLUT4 in skeletal muscle and adipose tissue. Unfortunately, patient 58D is
now deceased, and other family members were not available
for further analysis.
Type 2 diabetes usually results from a combination of
insulin resistance and impaired insulin secretion. Signaling
through IRS-1- and PI 3-kinase-dependent pathways is important not only for metabolic actions of insulin, but also for
normal ␤-cell function (52). Thus, it is possible that the T608R
Esposito et al. • Clinical Case Seminar
mutation in IRS-1 also contributed to ␤-cell defects and impaired insulin secretion in patient 58D, resulting in frank
diabetes. This would not be unprecedented, as the G972R
mutation in IRS-1 has also been implicated in both insulin
resistance and defective ␤-cell function (15, 20 –24). However,
it should be noted that studies analyzing the prevalence of
the G972R mutation in different populations have been controversial. In some studies the G972R mutation tended to
have an increased prevalence in diabetic patients compared
with normoglycemic individuals. However, these differences did not achieve statistical significance (9, 11, 37, 38, 53).
Other studies reported a similar prevalence of the G972R
mutation in diabetic patients and normoglycemic individuals (10, 13, 37, 39 – 43) or sometimes an even higher prevalence of the mutation in normoglycemic individuals (19, 38,
44). There are also a few reports that the G972R variant may
have an increased prevalence in subgroups of diabetic patients with more severe insulin resistance or dyslipidemia
and in patients with impaired glucose tolerance (19, 41). As
the T608R mutant is rare and has not been previously reported, it is not possible to meaningfully analyze the prevalence of this IRS-1 mutation in diabetic subjects at present.
In summary, we report a novel T608R mutation in IRS-1
detected in a patient with type 2 diabetes. This mutant has
an impaired ability to bind and activate PI 3-kinase and a
defect in mediating translocation of GLUT4 in adipose cells.
This mutation in IRS-1 may contribute to the pathogenesis of
diabetes in affected individuals by selectively impairing the
metabolic actions of insulin.
Acknowledgments
We thank patients, control subjects, and the son of patient 58D who
participated in the study.
Received June 14, 2002. Accepted January 21, 2003.
Address all correspondence and requests for reprints to: Michael J.
Quon, M.D., Ph.D., Diabetes Unit, Laboratory of Clinical Investigation,
National Center for Complementary and Alternative Medicine, National
Institutes of Health, Building 10, Room 8C-218, 10 Center Drive, MSC
1755, Bethesda, Maryland 20892-1755. E-mail: [email protected].
This work was supported by Telethon-Italy Grant E.0606 (to D.L.E.)
and by a grant from the Italian Ministry of Instruction, University and
Research to the Center of Excellence on Aging of the University of Chieti.
References
1. DeFronzo RA 1997 Pathogenesis of type 2 diabetes: metabolic and molecular
implications for identifying diabetes genes. Diabetes Rev 5:177–269
2. Gottlieb MS 1980 Diabetes in offspring and siblings of juvenile-and maturityonset type diabetics. J Chronic Dis 33:331–339
3. Harris MI 1998 Diabetes in America: epidemiology and scope of the problem.
Diabetes Care 21:C11–C14
4. Harris MI, Flegal KM, Cowie CC, Eberhardt MS, Goldstein DE, Little RR,
Wiedmeyer HM, Byrd-Holt DD 1998 Prevalence of diabetes, impaired fasting
glucose, and impaired glucose tolerance in U. S. adults. The Third National Health
and Nutrition Examination Survey 1988 –1994. Diabetes Care 21:518 –524
5. Poulsen P, Kyvik KO, Vaag A, Beck-Nielsen H 1999 Heritability of type II
(non-insulin-dependent) diabetes mellitus and abnormal glucose tolerance a
population-based twin study. Diabetologia 42:139 –145
6. Medici F, Hawa M, Ianari A, Pyke DA, Leslie RD 1999 Concordance rate for
type II diabetes mellitus in monozygotic twins: actuarial analysis. Diabetologia
42:146 –150
7. Taylor SI, Cama A, Accili D, Barbetti F, Quon MJ, de la Luz Sierra M, Suzuki
Y, Koller E, Levy-Toledano R, Wertheimer E, Moncada VY, Kadowaki H,
Kadowaki T 1992 Mutations in the insulin receptor gene. Endocr Rev 13:
566 –595
8. Bruning JC, Winnay J, Bonner-Weir S, Taylor SI, Accili D, Kahn CR 1997
Esposito et al. • Clinical Case Seminar
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
Development of a novel polygenic model of NIDDM in mice heterozygous for
IR and IRS-1 null alleles. Cell 88:561–572
Almind K, Bjorbaek C, Vestergaard H, Hansen T, Echwald S, Pedersen O
1993 Amino acid polymorphisms of insulin receptor substrate-1 in non-insulindependent diabetes mellitus. Lancet 342:828 – 832
Laakso M, Malkki M, Kekalainen P, Kuusisto J, Deeb SS 1994 Insulin
receptor substrate-1 variants in non-insulin-dependent diabetes. J Clin Invest
94:1141–1146
Imai Y, Fusco A, Suzuki Y, Lesniak MA, D’Alfonso R, Sesti G, Bertoli A,
Lauro R, Accili D, Taylor SI 1994 Variant sequences of insulin receptor
substrate-1 in patients with noninsulin-dependent diabetes mellitus. J Clin
Endocrinol Metab 79:1655–1658
Armstrong M, Haldane F, Taylor RW, Humphriss D, Berrish T, Stewart MW,
Turnbull DM, Alberti KG, Walker M 1996 Human insulin receptor substrate-1: variant sequences in familial non-insulin-dependent diabetes mellitus. Diabet Med 13:133–138
Ura S, Araki E, Kishikawa H, Shirotani T, Todaka M, Isami S, Shimoda S,
Yoshimura R, Matsuda K, Motoyoshi S, Miyamura N, Kahn CR, Shichiri M
1996 Molecular scanning of the insulin receptor substrate-1 (IRS-1) gene in
Japanese patients with NIDDM: identification of five novel polymorphisms.
Diabetologia 39:600 – 608
Esposito DL, Mammarella S, Ranieri A, Della Loggia F, Capani F, Consoli
A, Mariani-Costantini R, Caramia FG, Cama A, Battista P 1996 Deletion of
Gly723 in the insulin receptor substrate-1 of a patient with noninsulin-dependent diabetes mellitus. Hum Mutat 7:364 –366
Yoshimura R, Araki E, Ura S, Todaka M, Tsuruzoe K, Furukawa N, Motoshima H, Yoshizato K, Kaneko K, Matsuda K, Kishikawa H, Shichiri M 1997
Impact of natural IRS-1 mutations on insulin signals: mutations of IRS-1 in the
PTB domain and near SH2 protein binding sites result in impaired function at
different steps of IRS-1 signaling. Diabetes 46:929 –936
Zeng W, Peng J, Wan X, Chen S, Song H 2000 The mutation of insulin receptor
substrate-1 gene in Chinese patients with non-insulin-dependent diabetes
mellitus. Chin Med J 113:80 – 83
Celi FS, Negri C, Tanner K, Raben N, De Pablo F, Rovira A, Pallardo LF,
Martin-Vaquero P, Stern MP, Mitchell BD, Shuldiner AR 2000 Molecular
scanning for mutations in the insulin receptor substrate-1 (IRS-1) gene in
Mexican Americans with type 2 diabetes mellitus. Diabetes Metab Res Rev
16:370 –377
Whitehead JP, Humphreys P, Krook A, Jackson R, Hayward A, Lewis H, Siddle
K, O’Rahilly S 1998 Molecular scanning of the insulin receptor substrate 1 gene
in subjects with severe insulin resistance: detection and functional analysis of a
naturally occurring mutation in a YMXM motif. Diabetes 47:837– 839
Zhang Y, Wat N, Stratton IM, Warren-Perry MG, Orho M, Groop L, Turner
RC 1996 UKPDS 19: heterogeneity in NIDDM: separate contributions of IRS-1
and ␤3-adrenergic-receptor mutations to insulin resistance and obesity respectively with no evidence for glycogen synthase gene mutations. UK Prospective
Diabetes Study. Diabetologia 39:1505–11
Imai Y, Philippe N, Sesti G, Accili D, Taylor SI 1997 Expression of variant
forms of insulin receptor substrate-1 identified in patients with noninsulindependent diabetes mellitus. J Clin Endocrinol Metab 82:4201– 4207
Almind K, Inoue G, Pedersen O, Kahn CR 1996 A common amino acid
polymorphism in insulin receptor substrate-1 causes impaired insulin signaling. Evidence from transfection studies. J Clin Invest 97:2569 –2575
Hribal ML, Federici M, Porzio O, Lauro D, Borboni P, Accili D, Lauro R, Sesti
G 2000 The Gly3 Arg972 amino acid polymorphism in insulin receptor substrate-1 affects glucose metabolism in skeletal muscle cells. J Clin Endocrinol
Metab 85:2004 –2013
Porzio O, Federici M, Hribal ML, Lauro D, Accili D, Lauro R, Borboni P, Sesti
G 1999 The Gly9723 Arg amino acid polymorphism in IRS-1 impairs insulin
secretion in pancreatic beta cells. J Clin Invest 104:357–364
Federici M, Hribal ML, Ranalli M, Marselli L, Porzio O, Lauro D, Borboni
P, Lauro R, Marchetti P, Melino G, Sesti G 2001 The common Arg972 polymorphism in insulin receptor substrate-1 causes apoptosis of human pancreatic islets. FASEB J 15:22–24
Esposito DL, Li Y, Cama A, Quon MJ 2001 Tyr612 and Tyr632 in human insulin
receptor substrate-1 are important for full activation of insulin-stimulated
phosphatidylinositol 3-kinase activity and translocation of GLUT4 in adipose
cells. Endocrinology 142:2833–2840
Esposito DL, Palmirotta R, Verı̀ MC, Mammarella S, D’Amico F, Curia MC,
Aceto G, Crognale S, Creati B, Mariani-Costantini R, Battista P, Cama A 1998
Optimized PCR labeling in mutational and microsatellite analysis. Clin Chem
44:1381–1387
Araki E, Sun XJ, Haag BL, Chuang LM, Zhang Y, Yang-Feng TL, White MF,
Kahn CR 1993 Human skeletal muscle insulin receptor substrate-1. Characterization of the cDNA, gene, and chromosomal localization. Diabetes 42:1041–1054
Choi T, Huang M, Gorman C, Jaenisch R 1991 A generic intron increases gene
expression in transgenic mice. Mol Cell Biol 11:3070 –3074
Quon MJ, Zarnowski MJ, Guerre-Millo M, de la Luz Sierra M, Taylor SI,
Cushman SW 1993 Transfection of DNA into isolated rat adipose cells by
electroporation: evaluation of promoter activity in transfected adipose cells
which are highly responsive to insulin after one day in culture. Biochem
Biophys Res Commun 194:338 –346
J Clin Endocrinol Metab, April 2003, 88(4):1468 –1475 1475
30. Quon MJ, Butte AJ, Zarnowski MJ, Sesti G, Cushman SW, Taylor SI 1994 Insulin
receptor substrate 1 mediates the stimulatory effect of insulin on GLUT4 translocation in transfected rat adipose cells. J Biol Chem 269:27920 –27924
31. Quon MJ, Guerre-Millo M, Zarnowski MJ, Butte AJ, Em M, Cushman SW,
Taylor SI 1994 Tyrosine kinase-deficient mutant human insulin receptors
(Met11533 Ile) overexpressed in transfected rat adipose cells fail to mediate
translocation of epitope-tagged GLUT4. Proc Natl Acad Sci USA 91:5587–5591
32. Quon MJ, Cama A, Taylor SI 1992 Postbinding characterization of five naturally occurring mutations in the human insulin receptor gene: impaired
insulin-stimulated c-jun expression and thymidine incorporation despite normal receptor autophosphorylation. Biochemistry 31:9947–9954
33. Chen H, Wertheimer SJ, Lin CH, Katz SL, Amrein KE, Burn P, Quon MJ 1997
Protein-tyrosine phosphatases PTP1B and syp are modulators of insulin-stimulated translocation of GLUT4 in transfected rat adipose cells. J Biol Chem
272:8026 – 8031
34. Quon MJ, Chen H, Ing BL, Liu ML, Zarnowski MJ, Yonezawa K, Kasuga M,
Cushman SW, Taylor SI 1995 Roles of 1-phosphatidylinositol 3-kinase and ras
in regulating translocation of GLUT4 in transfected rat adipose cells. Mol Cell
Biol 15:5403–5411
35. Zhou L, Chen H, Lin CH, Cong LN, McGibbon MA, Sciacchitano S, Lesniak
MA, Quon MJ, Taylor SI 1997 Insulin receptor substrate-2 (IRS-2) can mediate
the action of insulin to stimulate translocation of GLUT4 to the cell surface in
rat adipose cells. J Biol Chem 272:29829 –29833
36. Zhou L, Chen H, Xu P, Cong LN, Sciacchitano S, Li Y, Graham D, Jacobs AR,
Taylor SI, Quon MJ 1999 Action of insulin receptor substrate-3 (IRS-3) and
IRS-4 to stimulate translocation of GLUT4 in rat adipose cells. Mol Endocrinol
13:505–514
37. Hitman GA, Hawrami K, McCarthy MI, Viswanathan M, Snehalatha C,
Ramachandran A, Tuomilehto J, Tuomilehto-Wolf E, Nissinen A, Pedersen
O 1995 Insulin receptor substrate-1 gene mutations in NIDDM; implications
for the study of polygenic disease. Diabetologia 38:481– 486
38. Hart LM, Stolk RP, Dekker JM, Nijpels G, Grobbee DE, Heine RJ, Maassen
JA 1999 Prevalence of variants in candidate genes for type 2 diabetes mellitus
in The Netherlands: the Rotterdam study and the Hoorn study. J Clin Endocrinol Metab 84:1002–1006
39. Mori H, Hashiramoto M, Kishimoto M, Kasuga M 1995 Amino acid polymorphisms of the insulin receptor substrate-1 in Japanese noninsulin-dependent diabetes mellitus. J Clin Endocrinol Metab 80:2822–2826
40. Sigal RJ, Doria A, Warram JH, Krolewski AS 1996 Codon 972 polymorphism
in the insulin receptor substrate-1 gene, obesity, and risk of noninsulin dependent diabetes mellitus. J Clin Endocrinol Metab 81:1657–1659
41. Yamada K, Yuan X, Ishiyama S, Shoji S, Kohno S, Koyama K, Koyanagi A,
Koyama W, Nonaka K 1998 Codon 972 polymorphism of the insulin receptor
substrate-1 gene in impaired glucose tolerance and late-onset NIDDM. Diabetes Care 21:753–756
42. Lei HH, Coresh J, Shuldiner AR, Boerwinkle E, Brancati FL 1999 Variants of
the insulin receptor substrate-1 and fatty acid binding protein 2 genes and the
risk of type 2 diabetes, obesity, and hyperinsulinemia in African-Americans:
the Atherosclerosis Risk in Communities Study. Diabetes 48:1868 –1872
43. Ito K, Katsuki A, Furuta M, Fujii M, Tsuchihashi K, Hori Y, Yano Y, Sumida
Y, Adachi Y 1999 Insulin sensitivity is not affected by mutation of codon 972
of the human IRS-1 gene. Horm Res 52:230 –234
44. Shimokawa K, Kadowaki H, Sakura H, Otabe S, Hagura R, Kosaka K, Yazaki
Y, Akanuma Y, Kadowaki T 1994 Molecular scanning of the glycogen synthase
and insulin receptor substrate-1 genes in Japanese subjects with non-insulin
dependent diabetes mellitus. Biochem Biophys Res Commun 202:463– 469
45. Sun XJ, Rothenberg P, Kahn CR, Backer JM, Araki E, Wilden PA, Cahill DA,
Goldstein BJ, White MF 1991 Structure of the insulin receptor substrate IRS-1
defines a unique signal transduction protein. Nature 352:73–77
46. Araki E, Haag III BL, Kahn CR 1994 Cloning of the mouse insulin receptor
substrate-1 (IRS-1) gene and complete sequence of mouse IRS-1. Biochim
Biophys Acta 1221:353–356
47. Wang L, Hayashi H, Mitani Y, Ishii K, Ohnishi T, Niwa Y, Kido H, Ebina
Y 1995 Cloning of a cDNA encoding a 190-kDa insulin receptor substrate-1-like
protein of simian COS cells. Biochem Biophys Res Commun 2:321–328
48. Taouis M, Taylor SI, Reitman M 1996 Cloning of the chicken insulin receptor
substrate 1 gene. Gene 178:51–55
49. Nystrom FH, Quon MJ 1999 Insulin signalling: metabolic pathways and mechanisms for specificity. Cell Signal 11:563–574
50. Backer JM, Myers Jr MG, Shoelson SE, Chin DJ, Sun XJ, Miralpeix M, Hu
P, Margolis B, Skolnik EY, Schlessinger J, White MF 1992 Phosphatidylinositol 3⬘-kinase is activated by association with IRS-1 during insulin stimulation.
EMBO J 11:3469 –3479
51. Rordorf-Nikolic T, Van Horn DJ, Chen D, White MF, Backer JM 1995 Regulation of phosphatidylinositol 3⬘-kinase by tyrosyl phosphoproteins. Full
activation requires occupancy of both SH2 domains in the 85-kDa regulatory
subunit. J Biol Chem 270:3662–3666
52. Burks DJ, White MF 2001 IRS proteins and ␤-cell function. Diabetes 50:S140 –S145
53. Hager J, Zouali H, Velho G, Froguel P 1993 Insulin receptor substrate (IRS-1)
gene polymorphisms in French NIDDM families. Lancet 342:1430