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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 4 1468-1475
Copyright © 2003 by The Endocrine Society

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

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: quonm{at}nih.gov.

Abstract

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 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 3-kinase 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 3-kinase-dependent pathways.

TYPE 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 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, 10, 11, 12, 13, 14, 15, 16, 17, 18). However, some of these are either rare IRS-1 variants (14, 15, 16, 17, 18) or low frequency polymorphisms that show similar prevalence in diabetic and control subjects (9, 10, 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, 21, 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 diabetes. 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 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 d. Lipofectamine Plus reagent (Life Technologies, Inc., Gaithersburg, MD) was then used to transiently transfect cells with 4 µg plasmid DNA/dish as described by the manufacturer.

Coimmunoprecipitation of p85 with recombinant IRS-1

NIH-3T3IR cells transiently transfected with pCIS2, IRS1-WT, or IRS1-T608R 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 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 (Bio-Rad Laboratories, Inc., 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. Protein A-Sepharose [50 µl 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{alpha} 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 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 PI (Sigma-Aldrich, St. Louis, MO) were sonicated in 10 µl PI 3-kinase reaction buffer [20 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 0.3 mM EGTA] and 10 µCi [{gamma}-32P]ATP in 40 µl PI 3-kinase reaction buffer (supplemented with 10 mM MgCl2) were added. The phosphorylation reaction was started by adding 50 µl of the substrate solution to 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 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 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 1Go. Among previously reported amino acid variants (9, 10, 11, 12, 13, 14, 15, 16, 17, 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 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. 1Go, 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. 1CGo). 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 resistance 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.


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  		Table 1. Allele frequencies of the IRS-1 variants detected

 


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  		Figure 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.

 
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. 2Go, 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. 2Go, 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. 3AGo, bottom panel, and Fig. 3DGo; P < 0.46). Coimmunoprecipitation of p85 was determined by immunoblotting samples with an anti-p85 antibody (Fig. 3AGo, 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. 3AGo, top panel, and Fig. 3BGo, 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. 3AGo, top panel, and Fig. 3BGo, 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. 3AGo, top panel, and Fig. 3BGo, 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.



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  		Figure 2. Insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1 is unaffected by the T608R IRS-1 mutation. 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. 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.

 


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  		Figure 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 [{gamma}-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{alpha} 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.

 
To determine whether decreased binding of p85 with IRS1-T608R 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. 3AGo, middle panel, and Fig. 3CGo). 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. 3AGo, middle panel, and Fig. 3CGo, 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. 3AGo, middle panel, and Fig. 3CGo, 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. 3AGo, middle panel, and Fig. 3CGo, 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. 4AGo, lower panel, and Fig. 4BGo; P < 0.005). However, in contrast to our results with p85 and PI 3-kinase activity, the amount of Grb-2 associated with IRS1-T608R in the insulin-stimulated state was similar to that observed for WT IRS-1. Moreover, as previously shown in Fig. 2Go, the amount of tyrosine-phosphorylated recombinant IRS-1 was comparable for WT and mutant IRS-1 (Fig. 4AGo, upper panel, and Fig. 4BGo; 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.



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  		Figure 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.

 
IRS1-T608R has an impaired ability to mediate translocation of GLUT4 in rat adipose cells

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. 5BGo). As expected, in control cells transfected with pCIS2 and GLUT4-HA, insulin stimulation resulted in a dose-dependent increase in GLUT4 at the cell surface (Fig. 5AGo). 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. 5AGo; 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.



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  		Figure 5. IRS1-T608R has an impaired ability to mediate translocation of GLUT4 in rat adipose cells. Adipose cells were cotransfected with GLUT4-HA (1 µg/cuvette) and pCIS2, WT IRS-1, or IRS1-T608R (4 µg/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 IRS1-T608R 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 HA-tagged recombinant IRS-1. Total membrane fractions derived from cells cotransfected with GLUT4-HA (1 µg/cuvette) and pCIS2, WT IRS-1, or IRS1-T608R (4 µg/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 GLUT4-HA. Representative blots are shown from an experiment that was repeated independently three times.

 
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, 38, 39, 40, 41, 42, 43, 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, 46, 47, 48), the nonconservative nature of the amino acid substitution, and the close proximity of position 608 to the PI 3-kinase 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 tyrosyl-phosphorylated 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 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 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 insulin-stimulated 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 insulin-stimulated 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 ß-cell function (52). Thus, it is possible that the T608R mutation in IRS-1 also contributed to ß-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 ß-cell function (15, 20, 21, 22, 23, 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, 40, 41, 42, 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.

Footnotes

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.

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.

Received June 14, 2002.

Accepted January 21, 2003.

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