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Original Studies |
Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health (Y.I., N.P., D.A., S.I.T.), Bethesda, Maryland 20892; and Dipartimento di Medicina Interna, Universita di Roma, "Tor Vergata" (G.S.), Rome, Italy
Address all correspondence and requests for reprints to: Simeon I. Taylor, M.D., Ph.D., National Institutes of Health, Building 10, Room 9S-213, 10 Center Drive, Bethesda, Maryland 20892. E-mail: Simeon_Taylor{at}nih.gov
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Arg,
Gly972Arg, and Arg1221Cys. We constructed
four IRS-1 expression vectors for transfection in COS-7 cells:
wild-type, single mutant (Gly819Arg), double mutant
(Gly819Arg; Gly972Arg), and triple mutant
(Gly819Arg; Gly972Arg;
Arg1221Cys) IRS-1. The mutations did not alter the level
of expression or the extent of insulin receptor-mediated tyrosine
phosphorylation of recombinant IRS-1. Moreover, the mutations did not
lead to a detectable impairment in the association of recombinant IRS-1
with important downstream effectors, including the p85 subunit of
phosphatidylinositol 3-kinase and growth factor receptor-binding
protein-2. We conclude that these amino acid substitutions do not appear to cause a major defect in the function of IRS-1, as judged by our assays. However, this type of assay probably lacks the sensitivity to detect subtle functional defects. In light of the suggestive associations observed in epidemiological studies, it is premature to totally discard the hypothesis that variant sequences of IRS-1 may contribute to the pathogenesis of NIDDM. Nevertheless, our studies cannot be interpreted as lending support to that hypothesis.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and -4 genes, and
mitochondrial DNA cause rare forms of NIDDM (6, 7, 8, 9, 10). Insulin receptor substrate-1 (IRS-1) is one of the substrates phosphorylated by the insulin receptor tyrosine kinase. Phosphorylation of one or more tyrosine residues in IRS-1 leads it to bind several proteins with SH2 domains (11): the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) (12), growth factor receptor-binding protein-2 (GRB-2) (13, 14), and SH2 domain-containing phosphotyrosine phosphatase (15). The coupling of IRS-1 with these signaling molecules via their SH2 domains is hypothesized to activate other effector proteins that are important for insulin action. IRS-1 knockout mice have revealed that IRS-1 is important for both mitogenesis and metabolic response; mice lacking IRS-1 exhibit growth retardation and insulin resistance (16, 17).
In light of the role of IRS-1 in insulin action, mutations in the IRS-1 gene might contribute to the insulin resistance observed in patients with NIDDM. This hypothesis has motivated several laboratories to screen for mutations in the IRS-1 gene of patients with NIDDM (18, 19, 20, 21, 22). There are several variant forms of IRS-1 originally identified in patients with NIDDM. However, the pathological significance of these variant sequences is controversial because most of these variants have been found in both diabetic and normal subjects. Nevertheless, some studies have reported an increase in the prevalence of variant forms of IRS-1 in patients with NIDDM, suggesting a pathogenic role for the amino acid substitutions (18, 19, 20, 22). Therefore, in this study we have overexpressed several variant forms of IRS-1 in COS-7 cells to investigate directly whether the amino acid substitutions impair the function of IRS-1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Antiphosphotyrosine antibody (mouse monoclonal), antirat carboxy-terminal IRS-1 antibody (rabbit polyclonal), antirat PI 3-kinase antibody (rabbit polyclonal), and anti-GRB-2 antibody (mouse monoclonal) were obtained from Upstate Biotechnology (Lake Placid, NY). Mammalian expression vector pCMV-human insulin receptor was described previously (23). Human recombinant insulin was obtained from Sigma Chemical Co. (St. Louis, MO).
Construction of human IRS-1 expression vector with variant sequences
We identified a patient with NIDDM who was heterozygous for
three amino acids substitutions of IRS-1: Gly819Arg
(G819R), Gly972Arg (G972R), and
Arg1221Cys (R1221C) (20). The family of the patient was
screened for these amino acid substitutions. The spouse also had NIDDM
and was heterozygous for the G972R substitution. The two children
appeared healthy. Although the daughter was heterozygous for the G972R
substitution, the son did not carry any of three substitutions. Because
the son did not inherit any of the polymorphic sequences, this suggests
that all three substitutions are on the same allele in the parent with
three variant sequences. Therefore, we have constructed three IRS-1
expression vectors with the following variant sequences: G972R,
G819R-G972R, and G819R-G972R-R1221C. A fragment of human genomic DNA
containing the complete coding sequence of IRS-1 was cloned and ligated
into pGEM-4Z (Promega, Madison, WI). The sequence of the entire coding
region was determined and was identical to the sequence reported by
Araki et al. (24). Mutagenesis of human IRS-1 was performed
as follows. PCR was performed using as template genomic DNA from a
patient who is heterozygous for the G972R substitution. The PCR
fragment was digested with BamHI and NheI
restriction endonucleases and ligated into pGEM-4Z-wild-type IRS-1
(pGEM-4Z-WT-IRS-1) that had previously been digested with the same
enzymes. The method of Higuchi et al. (25) was used to
introduce the other variant sequences into IRS-1 complementary DNA
(cDNA). The cDNAs encoding WT-IRS-1, G972R-IRS-1, G819R-G972R-IRS-1,
and G819R-G972R-R1221C-IRS-1 were then ligated into pcDNA3 mammalian
expression vector (Invitrogen Corp., San Diego, CA).
Expression of IRS-1 in COS-7 cells and in vivo tyrosine phosphorylation of IRS-1
COS-7 cells were cultivated in DMEM with 10% FBS. One day before the experiment, cells were plated in 100 x 20-mm dishes at a density of 5 x 105 cells/dish. Each dish was treated with transfection medium containing 1.6 µg pcDNA3-IRS-1 and 0.2 µg pCMV-human insulin receptor suspended with 64 µL Lipofectamine (Life Technologies, Gaithersburg, MD) in Opti-MEM (Life Technologies). After transfection was carried out for 5 h at 37 C, the media were changed to DMEM with 10% FBS, and cells were incubated for 21 h in a humidified incubator. After this incubation, cells were starved for 3 h in DMEM with 1% insulin-free BSA (Intergen Co., Purchase, NY). Thereafter, cells were stimulated with insulin (0–100 nmol/L) for 2 min, frozen in liquid nitrogen, and solubilized in 0.5 mL lysis buffer [1% Nonidet P-40 (NP-40), 150 mmol/L NaCl, 50 mmol/L HEPES (pH 7.6), 1 mmol/L MgCl2, 1 mmol/L CaCl2, 10% glycerol, 100 mmol/L sodium fluoride, 10 mmol/L sodium pyrophosphate, 2 mmol/L sodium vanadate, 0.3 µg/mL phenylmethylsulfonylfluoride, 1 µg/mL pepstatin A, 1 µg/mL aprotinin, 10 µg/mL chymostatin, 10 µg/mL antipain-dihydrochloride, and 1 µg/mL leupeptin]. Insoluble material was removed by centrifugation at 13,000 x g for 10 min, and the supernatant was saved for analysis by immunoprecipitation and immunoblotting.
Immunoprecipitation
Cell lysate (0.5 mL) was incubated with 4 µg anti-IRS-1 antibody overnight at 4 C. Immune complexes were collected by incubation with immobilized protein A (Pierce Chemical Co., Rockford, IL) at 4 C for 2 h. Immobilized protein A was sedimented by centrifugation at 7000 x g for 1 min, washed three times with lysis buffer, and resuspended in 2 x Laemmli sample buffer (26).
Immunoblotting
Cell lysate or immunoprecipitated protein was separated by SDS-PAGE and transferred to a polyvinylidine difluoride (PVDF) membrane using the Xcell II Mini-cell apparatus (Novex, San Diego CA). The blot was placed into 10 mmol/L Tris (pH 7.5) and 150 mmol/L NaCl (TBS) containing 0.2% NP-40 and 3% BSA (blocking buffer) for 30 min at room temperature and incubated with the indicated antibodies in blocking buffer overnight at 4 C. Thereafter, the blot was washed three times for 15 min each time in TBS containing 1% NP-40 and once in TBS. Subsequently, the blot was incubated with horseradish peroxidase-conjugated anti-IgG antibodies in blocking buffer for 1 h at room temperature. The blot was again washed three times for 15 min each time in TBS containing 1% NP-40 and twice in TBS. Proteins were detected by treating the blot with enhanced chemiluminescence (SuperSignal CL-HRP Substrate System, Pierce) and exposing it to Kodak X-AR film (Eastman Kodak, Rochester, NY). Films were quantitated on a Molecular Dynamics densitometer (Molecular Dynamics, Sunnyvale, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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COS-7 cells were transiently cotransfected with expression vectors
for the human insulin receptor and various forms of human IRS-1
(WT-IRS-1, G972R-IRS-1, G819R-G972R-IRS-1, or
G819R-G972R-R1221C-IRS-1). Transfected cells were stimulated with
insulin (0–100 nmol/L) for 2 min and lysed. The lysate was analyzed by
immunoblotting using antiphosphotyrosine or anti-IRS-1 antibodies as
probes. The amino acid substitutions did not significantly alter the
level of recombinant IRS-1 protein expressed in the transfected cells
(Fig. 1
and data not shown). We have not
directly measured the stability of recombinant IRS-1. However, because
it is unlikely that a point mutation would alter the rate of
transcription or translation in this type of transient transfection
system, the fact that the steady state level of IRS-1 in our system was
equivalent suggests that the stability of the mutant proteins is
normal. Insulin increased tyrosine phosphorylation of both WT-IRS-1 and
variant IRS-1 in a dose-dependent manner (Figs. 1 and 2). Quantitation of immunoreactive bands
by densitometry showed that insulin (100 nmol/L) increased
phosphorylation of WT-IRS-1 3- to 5-fold over the basal level (Fig. 2).
The extent of IRS-1 phosphorylation was similar in variant IRS-1 and
WT-IRS-1 (Fig. 2). Thus, there was no major defect in tyrosine
phosphorylation of the three variant forms of IRS-1 compared with that
of WT-IRS-1. In control experiments, we transfected COS-7 cells with an
empty expression vector lacking the cDNA encoding IRS-1 (Fig. 3, lanes 1, 2, 7, and 8). Endogenous
IRS-1 was not detected in immunoblots probed with antibodies directed
against phosphotyrosine (-P-Tyr) or IRS-1 (-IRS-1) in these
cells. These observations confirm that endogenous levels of IRS-1 are
low in COS-7 cells, and that the majority of IRS-1 detected in our
assay is recombinant protein whose expression is driven by the
expression vector.
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First, we overexpressed IRS-1 (WT-IRS-1 and three mutant IRS-1) in
COS-7 cells that were not cotransfected with insulin receptor
expression vector. Cells were incubated in the presence or absence of
insulin (100 nmol/L) and lysed. After immunoprecipitation of cell
lysates with antibody to IRS-1, the amount of p85 subunit of PI
3-kinase associated with IRS-1 was determined by densitometric analysis
of immunoblots probed with anti-p85 antibody (Fig. 3
, left
half). Insulin increased the association of p85 subunit with
WT-IRS-1 about 4-fold over the basal level (Figs. 3 and 4). The association of p85 subunit with
IRS-1 was similar in WT-IRS-1 and all three variant molecules (Fig. 4).
Using antiphosphotyrosine immunoblotting, we did not detect a band
corresponding to phosphorylated IRS-1 in cells that were not
co-transfected with expression vector for the insulin receptor.
Nevertheless, the observation that insulin stimulated association of
p85 with IRS-1 provides indirect evidence that insulin stimulated
tyrosine phosphorylation of IRS-1 under these experimental conditions,
although the antiphosphotyrosine blotting techniques appeared to lack
sufficient sensitivity to detect this low level of phosphorylation.
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, right half). It is likely that the presence of a
vast excess of recombinant insulin receptors resulted in sufficient
tyrosine kinase activity to phosphorylate enough IRS-1 to promote
maximal association with the p85 subunit even in the absence of
insulin. We next carried out comparative studies in cells coexpressing
insulin receptors and the various forms of IRS-1. There was a slight
decrease in the amount of PI 3-kinase associated with G972R-IRS-1
compared with that of WT-IRS-1. However, there was no statistically
significant difference observed between WT-IRS-1 and G819R-G972R-IRS-1
or G819R-G972R-R1221C-IRS-1, even though these mutants also carry the
G972R substitution (Table 1). In summary, we did not
detect a reproducible effect of any of these three amino acid
substitutions to alter the ability of insulin or insulin receptors to
promote the association between IRS-1 and p85 subunit.
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). These data differ from
the experiments presented in Figs. 1 and 2, in which insulin did induce
an increase in the phosphotyrosine content of recombinant IRS-1.
Although it is not certain what led to the difference between the
observations in the two experiments, it is probably related to
differences in the transfection efficiencies and the levels of
expression of recombinant insulin receptors in the two sets of
experiments. Association of GRB-2 with WT-IRS-1 and mutant IRS-1
We used a similar experimental design to study the ability of
insulin to promote the association between IRS-1 and GRB-2. In cells
that overexpress IRS-1 in the absence of recombinant insulin receptors,
we had difficulty detecting the association between GRB-2 and IRS-1
(Fig. 3, left half). In contrast, coexpression of insulin
receptors markedly increased the quantity of GRB-2 that was
coimmunoprecipitated with IRS-1. However, we did not detect a
reproducible effect of insulin to promote the association between GRB-2
and IRS-1 under our experimental conditions (Fig. 4). In any case, we
compared the amount of GRB-2 associated with WT-IRS-1 and the three
variant forms of IRS-1 in COS-7 cells overexpressing both IRS-1 and
insulin receptor. However, we did not detect a statistically
significant difference between WT-IRS-1 and variant forms of IRS-1 in
the ability to associate with GRB-2 (Fig. 3 and data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A large body of evidence suggests that genetic factors predispose to the development of NIDDM (3). Accordingly, considerable effort has been devoted to identify genes that contribute to this genetic predisposition. In one approach to this question, several laboratories have screened for the presence of mutations in candidate genes that encode proteins with important functions in the pathways of insulin action (e.g. the insulin receptor) (5) or insulin secretion (e.g. glucokinase) (7). When variation is identified in the nucleotide sequence of a candidate gene, it is necessary to determine whether the variation is significant in the sense that it contributes to the pathogenesis of diabetes. At least three approaches have been taken to address this question: genetic linkage, genetic association, and biochemical studies. However, mutations have not been commonly found in the genes encoding insulin, the insulin receptor, or glucokinase. Thus, the genetic basis of the common form of NIDDM has not yet been identified. Nevertheless, the gene encoding IRS-1 is among the candidate genes that have been screened in the hope of identifying genetic defects in the pathway of insulin action. While the initial reports presented data that there is an association between NIDDM and amino acid sequence polymorphisms (especially G972R) in the IRS-1 gene (18, 20, 22), this finding has not been confirmed in several other studies (19, 21). Furthermore, genetic linkage studies have not provided evidence to support the hypothesis that mutations in the IRS-1 gene predispose to the development of NIDDM (27). Nevertheless, it remains possible that genetic and epidemiologic approaches might lack the statistical power to identify the role of a gene as a cause of NIDDM. For example, if NIDDM displays genetic heterogeneity, and polymorphisms in the IRS-1 gene contributed to the development of NIDDM in only a subpopulation of patients with NIDDM, this would make it more difficult to design a study that demonstrated this effect. The smaller the subpopulation, the more difficult it would be to demonstrate the effect. In addition, if the penetrance of the NIDDM phenotype were less than 100%, this would make it more difficult to demonstrate an effect of the IRS-1 gene.
Evaluation of the function of variant IRS-1 molecules
Because of the limitations inherent in the genetic and epidemiologic approaches to evaluate the significance of genetic variation at the IRS-1 locus, we have elected to supplement these approaches with an alternative strategy to address the same question. Accordingly, we have inquired whether the observed amino acid substitutions impair the function of IRS-1. In the present studies, none of the three amino acid substitutions (i.e. G819R, G972R, and R1221C) led to a detectable defect in the function of the IRS-1 molecule under our experimental conditions. In particular, we did not detect any abnormality in the stability or the phosphorylation of IRS-1. Neither did we detect any abnormality in the ability of variant forms of IRS-1 to bind SH2 domain-containing proteins, such as the p85 subunit of PI 3-kinase or GRB-2. It is likely that our studies would have detected a major abnormality in any of these functions of the IRS-1 function. Nevertheless, we cannot rule out the possibility that there might be a partial functional defect that was too subtle to be detected by our experimental approach. For example, because of the presence of multiple phosphorylation sites in the IRS-1 molecule, measurement of total phosphotyrosine content is not a sensitive method to detect a selective defect in the phosphorylation of a single tyrosine residue. Similarly, the degree of variability in quantitating the binding of other molecules (e.g. the p85 subunit of PI 3-kinase) makes it difficult to rule out the possibility of a partial decrease in the affinity of IRS-1 to bind the SH2 domain(s). Furthermore, although it is likely that there are other unknown SH2 domain-containing proteins that bind to IRS-1, we have investigated the binding of only two proteins (i.e. the p85 subunit of PI 3-kinase and GRB-2 to IRS-1. Nevertheless, as discussed below, there are several reasons to predict that it would require a major functional defect for a heterozygous mutation in IRS-1 to impair insulin action.
After our work was completed, Almind et al. (28) reported
that the G972R mutation causes a 25% decrease in the binding of the
p85 regulatory subunit of PI 3-kinase. In association with the impaired
binding of IRS-1 to p85, they observed a 36% decrease in the PI
3-kinase activity coimmunoprecipitated with IRS-1 and a 32% decrease
in the mitogenic activity of insulin. Similar results were reported by
Yoshimura et al. (29). Our data appear to contradict these
other studies in that we disagree about whether there is a subtle
functional defect (
25–35%) in the ability of IRS-1 to activate PI
3-kinase. However, all three studies agree that the G972R substitution
does not lead to a large quantitative defect in the function of IRS-1.
If one assumes that this amino acid substitution is present in only one
allele of the IRS-1 gene, then heterozygosity would be predicted to
cause only about a 15% decrease in IRS-1 function within the cell. It
is difficult to assess whether such a small defect might be important
from a physiological point of view.
Animal model for null mutations in the IRS-1 gene
Mutant mice have been obtained in which the IRS-1 gene has been inactivated by homologous recombination (16, 17). In the homozygous state, the null allele of the IRS-1 gene markedly impairs growth and leads to insulin resistance. Nevertheless, insulin is capable of eliciting a biological response even in mice that totally lack IRS-1. A homologous protein (i.e. IRS-2) has been identified (30). It has been proposed that IRS-2 provides an alternate pathway that may be responsible for replacing the function of IRS-1 in the IRS-1 knock-out mice (17). These observations in mice raise the following question. Is it plausible to propose that heterozygosity for a variant allele of the human IRS-1 gene would cause insulin resistance? Of course, it is possible that there are species differences such that humans might be more sensitive than mice to the effects of a genetic defect in IRS-1 function. Recent evidence suggests that genetic variation at other loci may modulate the effects of a mutation at the IRS-1 locus. For example, as the result of breeding experiments, it was possible to obtain mice that were homozygous for mutations in the IRS-1 gene and the insulin receptor gene (31). These two mutations at different genetic loci appeared to have a synergistic effect to cause insulin resistance. Thus, it is possible that a mutation in the human IRS-1 gene would predispose to the development of insulin resistance and/or NIDDM only when present in individuals who also possess susceptibility alleles at other genetic loci. Nevertheless, in the mouse model, the mutation in the IRS-1 gene is a null mutation that completely abolishes the function of product encoded by that allele. Thus, it seems likely that a mutation would need to cause a major defect in the function of IRS-1 if it were to cause insulin resistance inherited in a genetically dominant pattern. However, at least under our experimental conditions, these three naturally occurring amino acid substitutions in the IRS-1 molecule did not cause a major defect in the function of IRS-1.
Interactions between obesity and variant IRS-1
Clausen et al. (32) provided evidence suggesting
that obesity and the G972R allele of IRS-1 act synergistically to cause
insulin resistance. According to their data, the G972R allele is not
associated with insulin resistance in lean individuals. However, when
the study was restricted to obese individuals, insulin resistance was
significantly more severe among heterozygous carriers of the G972R
allele than among individuals who are homozygous for the common allele
(i.e. encoding a glycine residue at position 972). These
observations suggest that the G972R variant IRS-1 molecule may somehow
facilitate the ability of obesity to cause insulin resistance. Recent
studies have suggested one possible molecular mechanism that might
explain these observations. It has been proposed that the ability of
adipose tissue to secrete tumor necrosis factor- (TNF) may
explain the fact that obesity leads to the development of insulin
resistance (33). Furthermore, TNF stimulates the phosphorylation of
serine and threonine residues in IRS-1, which, in turn, inhibits the
ability of the insulin receptor to phosphorylate tyrosine residues in
IRS-1 (33, 34). In brief, according to these hypotheses, the IRS-1
molecule participates in the pathway by which TNF causes insulin
resistance. It is reasonable to speculate that a variant IRS-1 molecule
might exert a dominant effect to cause insulin resistance if the amino
acid substitution rendered the molecule more susceptible to
TNF-induced serine/threonine phosphorylation.
In conclusion, three naturally occurring amino acid substitutions in human IRS-1 (i.e. G819R, G972R, and R1221C) do not appear to cause major defects in the function of IRS-1 as judged by our assays. Nevertheless, we have not totally ruled out the possibility that variant forms of IRS-1 have minor defects that were not detected by our assays and contribute to the phenotype of NIDDM in combination with other risk factors. However, our observations do not provide additional support for the hypothesis that variant sequences of IRS-1 act as a dominant genetic factor predisposing to the development of NIDDM.
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Acknowledgments |
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Footnotes |
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Received November 18, 1996.
Revised July 17, 1997.
Accepted August 13, 1997.
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References |
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