Human insulin-receptor gene. Partial sequence and amplification of exons by polymerase chain reaction

The partial sequence of the human insulin-receptor (hINSR) gene is presented. Using the gene sequence as a guide, we selected pairs of oligonucleotide primers from sites in the introns that flank each exon. These primers allowed each of the 22 exons of the hINSR gene to be amplified in vitro by the polymerase chain reaction. The sequences of the gene and oligonucleotide primers will facilitate studies of genetic variation in the hINSR gene and thereby increase our understanding of the role of this gene in the development of insulin-resistant states and glucose intolerance.     

Linkage analysis of insulin-receptor gene in familial NIDDM.

Although non-insulin-dependent diabetes mellitus (NIDDM) is clearly inherited, the mode of inheritance and genetic etiology remain unknown. Impaired insulin action is an important component of NIDDM, which may precede NIDDM onset, and appears to be inherited. Numerous defects of the insulin-receptor gene have been described in syndromes of extreme insulin resistance, and this gene is a strong candidate for genetic predisposition to NIDDM. To test this hypothesis, we examined 18 white pedigrees from Utah that had two or more siblings with NIDDM. For each pedigree, individuals not known to be affected were tested by standard oral glucose tolerance test, and diagnoses of NIDDM and impaired glucose tolerance were made by World Health Organization criteria. Each individual was typed for seven restriction-fragment-length polymorphism markers at the insulin-receptor locus, and marker phase was established by segregation. Linkage was examined with the LINKAGE program under six models, including autosomal dominant and autosomal recessive, with individuals with impaired glucose tolerance counted either as affected or of unknown status and with or without sporadic cases of diabetes. Under each model, linkage was significantly rejected. Neither inspection of individual pedigree log of odds scores nor formal tests of heterogeneity suggested a subgroup in which linkage of NIDDM and insulin-receptor gene was likely. In addition, sharing of insulin-receptor gene haplotypes among 108 affected sibling pairs drawn from the pedigrees did not deviate from that expected by chance alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Restriction-fragment-length polymorphism in insulin-receptor gene and insulin resistance in NIDDM

Restriction-enzyme analysis of genomic DNA from 52 White and Hispanic nondiabetic subjects and 51 subjects with non-insulin-dependent diabetes (NIDDM) was carried out with insulin-receptor cDNA probes. A polymorphic 5.8-kilobase SstI fragment was found in 12 (23.5%) of 51 NIDDM subjects but only in 4 (7.7%) of 52 nondiabetic control subjects. This association is significant by chi 2-analysis (P less than .05). Furthermore, the nondiabetic subjects with the polymorphism were found to have hyperinsulinemia and/or nondiagnostic glucose tolerance. The polymorphism is a genetic marker for a phenotype that is neither necessary nor, by itself, sufficient for NIDDM. Nevertheless, it may indicate that insulin resistance functionally related to an insulin-receptor gene polymorphism is the proximal cause of NIDDM in at least one subset of the population.     

Insulin-receptor cDNA sequence in NIDDM patient homozygous for insulin-receptor gene RFLP.

Resistance to insulin action is a well-established feature of non-insulin-dependent diabetes mellitus (NIDDM) and is believed to contribute to the etiology of this condition. A strong genetic contribution to the etiology of NIDDM exists, and we previously identified an insulin-receptor gene restriction-fragment-length polymorphism (RFLP) associated with the NIDDM phenotype. In an attempt to elucidate whether structural defects in the insulin receptor could be a primary cause of insulin resistance in NIDDM, we analyzed the insulin-receptor cDNA sequence in a subject with NIDDM who is also homozygous for this RFLP. The insulin-receptor cDNA was sequenced with the polymerase chain reaction (PCR). mRNA from transformed lymphocytes was reverse transcribed and amplified with five overlapping sets of primers that span the coding sequence of both alpha- and beta-subunits. No difference was found in the predicted amino acid sequence of the subject's insulin receptor compared with the normal insulin receptor. At nucleotide positions 831 and 2247, the subject is heterozygous for silent nucleotide polymorphisms that do not affect the amino acid sequence. Exon 11 encodes a 12-amino acid insert in the alpha-subunit, which, due to alternate splicing, is not expressed in lymphocyte insulin-receptor mRNA. Consequently, exon 11 was amplified from genomic DNA by PCR; the sequence of exon 11 was found to be normal. In addition, when this patient's transformed lymphocytes were maintained in culture, no abnormalities in insulin binding were observed. We conclude that the insulin resistance seen in this NIDDM subject is not due to a structural alteration in the insulin receptor itself.(ABSTRACT TRUNCATED AT 250 WORDS)     

Type II diabetes mellitus and polymorphism of insulin-receptor gene in Mexican Americans

Resistance to insulin action is a well-established feature of type II (non-insulin-dependent) diabetes and is believed by many to contribute to the etiology of this condition. We therefore characterized restriction-fragment-length polymorphisms of the insulin-receptor gene with the restriction enzyme Rsa 1 in 242 Mexican Americans and non-Hispanic Whites with type II diabetes and 202 age-, sex-, and ethnicity-matched control subjects who participated in a population-based study in San Antonio. Alleles of 6.7 kilobases (kb) (A allele), 6.2 kb (B allele), and 3.4 kb (C allele) were identified. The C allele was observed in Mexican Americans only, where its frequency among nondiabetic control subjects was 17.7%. Diabetic Mexican Americans were twice as likely as control subjects to be homozygous for the C allele. The crude odds ratio for diabetes in CC homozygotes compared with the other two genotypes was 2.22, although this result was not statistically significant (chi 2 = 1.57, P = .21). The Mantel-Haenszel odds ratio, adjusting for age, however, indicated a 4.71-fold increased risk of diabetes among Mexican Americans with the CC genotype compared with Mexican Americans without this genotype (chi 2 = 5.38, P = .020). The age of onset of diabetes was also slightly younger in CC homozygote cases (45.4 +/- 9.2 yr) than in CX or XX cases (47.7 +/- 9.0 and 48.6 +/- 9.6 yr, respectively), although this difference was not statistically significant (P .467).(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular and clinical characterization of an insertional polymorphism of the insulin-receptor gene

A restriction-fragment-length polymorphism (RFLP) detected with the human insulin-receptor cDNA and the enzyme Sac I has been reported to be associated with non-insulin-dependent diabetes mellitus (NIDDM) in White and Black populations and segregated with diabetes in two small pedigrees with maturity-onset diabetes of the young. A size difference of approximately 500 base pairs (bp) was demonstrated between the alleles of this and several other RFLPs that mapped to the same 300-bp region near the transmembrane coding region of the cDNA beta-chain, thus suggesting the presence of an insertion in this region that could affect insulin-receptor function. Genomic DNA fragments containing this RFLP were cloned from an individual heterozygous for the putative insertion, and the differing fragments of the two alleles were sequenced. The presence of a 400-bp insertion was thus confirmed and was demonstrated to be entirely within an intron. No significant coding-region differences from published cDNA sequences were detected in four exons sequenced from the region of the insertional allele. The sequenced regions included multiple Alu repeat sequences. The RFLP was unusual in that the larger allele consisted of an additional Alu repeat sequence that included a new Pst I site. Because the nature and location of the insertion did not suggest a role in insulin-receptor function, the association of this RFLP with NIDDM and hyperinsulinemia was reexamined in a small sample of Whites. No association could be demonstrated, and the insertion also failed to segregate with NIDDM in five White pedigrees.(ABSTRACT TRUNCATED AT 250 WORDS)     

Allelic variants at insulin-receptor and insulin gene loci and susceptibility to NIDDM in Welsh population

A cohort of 132 well-documented White Welsh non-insulin-dependent diabetic (NIDDM) subjects were genotyped for 5 restriction-fragment-length polymorphisms (RFLPs) at the insulin-receptor gene (IRG) locus and a polymorphic locus 5' to the insulin gene. There was no significant difference in RFLP frequencies between the NIDDM subjects and a group of 87 matched White control subjects. Paired haplotype analysis of the IRG RFLPs suggested a difference between NIDDM and control groups for the endonuclease combinations Bgl II-Rsa I and Bgl II-Xba I. Analysis of implied haplotypes defined by the endonucleases Bgl II, Rsa I, and Xba I revealed one haplotype to be more prevalent in the NIDDM group; whereas, another haplotype was associated with the control group (P less than 0.02). Subset analysis within the NIDDM cohort compared the metabolic response of NIDDM subjects with the differing IRG haplotypes to a standard meal tolerance test. Both groups showed equivalent basal and postprandial glucose excursions, but one group revealed a significantly exaggerated plasma insulin response compared with the other (P less than 0.05). This may reflect the influence of genetic variation at the IRG locus on insulin sensitivity in patients with NIDDM.     

Detection of mutations in insulin-receptor gene in NIDDM patients by analysis of single-stranded conformation polymorphisms

We used the recently described technique of single-stranded conformation-polymorphism (SSCP) analysis to examine the insulin-receptor locus. First, the ability of the method to detect known mutations and polymorphisms in the insulin-receptor coding sequence was assessed. Regions of the insulin-receptor sequence containing 16 different nucleotide changes, 9 in patient genomic DNA and 7 as cloned cDNA in plasmids, were analyzed. All 9 patient genomic DNA mutants and 5 of 7 plasmid mutants exhibited variant SSCP patterns. To investigate the potential of the technique for screening many patients, the 5 exons that encode the tyrosine kinase domain of the insulin receptor were examined in 30 unrelated white subjects with non-insulin-dependent diabetes mellitus (NIDDM). Exons 17-21 were amplified from genomic DNA with polymerase chain reaction and subjected to SSCP analysis. Exons 19, 20, and 21 revealed no bands of aberrant migration, suggesting a high degree of conservation of these sequences. One diabetic subject had an SSCP variant in exon 18. Direct sequencing of this subject's genomic DNA revealed a heterozygous missense mutation (Lys1068----Glu1068). Five different SSCP patterns were detected in exon 17. Based on direct sequencing, these patterns were explained by combinations of three different nucleotide substitutions, two of which were common silent polymorphisms. One subject had a heterozygous missense mutation Val985---- Met985. Allele-specific oligonucleotide hybridization confirmed the presence of these mutations in the appropriate diabetic subjects and also detected the Val985 mutation in heterozygous form in 1 of 13 nondiabetic white subjects. SSCP analysis is a sensitive rapid method for screening for mutations in the insulin-receptor gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of insulin-receptor mRNA levels by glucocorticoids

We found with IM-9 human cultured lymphocytes, that the glucocorticoid dexamethasone increased insulin-receptor mRNA levels. This increase correlated in a time- and dose-dependent manner with the increase in the biosynthesis of the insulin-receptor precursor. In addition, in AR42J cultured rat pancreatic acinar cells, dexamethasone increased insulin-receptor mRNA levels. These studies suggest, therefore, that an increase in mRNA levels is an early step in the regulation of the insulin receptor by glucocorticoids in several cell types.     

Alteration of insulin-receptor kinase activity by high-fat feeding

It has been demonstrated in in vivo and in vitro experiments that high-fat (HF) feeding causes insulin resistance. To elucidate the mechanism for this effect, we have measured the kinase activity of the insulin receptor purified from livers of HF-fed rats that showed impaired insulin action in isolated rat adipocytes. In adipocyte experiments, HF feeding led to a 65% decrease in the maximal response stimulated by insulin in a 2-deoxyglucose uptake study. Although insulin binding to adipocytes of HF-fed rats also decreased to 50% of control due to decreased binding affinity, the postbinding defect should be accounted for by decreased insulin action in view of the presence of spare receptor. In contrast to adipocytes, insulin binding to the lectin-purified insulin receptor from livers showed no difference in receptor-binding affinity between HF-fed and control rats. Insulin-stimulated phosphorylation of the beta-subunit of the insulin receptor was decreased to almost 50% throughout the entire dose-response curve. The study of glutamine-tyrosine (4:1) phosphorylation by the insulin-receptor kinase showed results similar to those of the autophosphorylation study. These results suggest that an HF diet causes insulin resistance by affecting insulin-receptor kinase, which plays an important role in transmembrane signaling between insulin binding and insulin action.     

Lilly Lecture: molecular mechanisms of insulin resistance. Lessons from patients with mutations in the insulin-receptor gene.

Insulin resistance contributes to the pathogenesis of NIDDM. We have investigated the molecular mechanisms of insulin resistance in patients with genetic syndromes caused by mutations in the insulin-receptor gene. In general, patients with two mutant alleles of the insulin-receptor gene are more severely insulin-resistant than are patients who are heterozygous for a single mutant allele. These mutations can be put into five classes, depending upon the mechanisms by which they impair receptor function. Some mutations lead to a decrease in the number of insulin receptors on the cell surface. For example, some mutations decrease the level of insulin receptor mRNA or impair receptor biosynthesis by introducing a premature chain termination codon (class 1). Class 2 mutations impair the transport of receptors through the endoplasmic reticulum and Golgi apparatus to the plasma membrane. Mutations that accelerate the rate of receptor degradation (class 5) also decrease the number of receptors on the cell surface. Other mutations cause insulin resistance by impairing receptor function--either by decreasing the affinity to bind insulin (class 3) or by impairing receptor tyrosine kinase activity (class 4). The prevalence of mutations in the insulin receptor gene is not known. However, theoretical calculations suggest that approximately 0.1-1% of the general population are heterozygous for a mutation in the insulin-receptor gene; the prevalence is likely to be higher among people with NIDDM. Accordingly, it is likely that mutations in the insulin-receptor gene may be a contributory cause of insulin resistance in a subpopulation with NIDDM.     

Normal insulin-receptor cDNA sequence in Pima Indians with NIDDM

Pima Indians have served as a model of non-insulin-dependent diabetes mellitus (NIDDM). Within this population, inherited insulin resistance is a primary determinant of abnormal glucose metabolism. The insulin receptor is regarded as a "candidate gene" that could potentially be defective in Pima Indians or other populations with NIDDM. To directly address the question of potential insulin-receptor genetic defects in Pima Indians, we isolated and sequenced insulin-receptor cDNA from two Pima Indians with NIDDM. Small amounts of lymphoblast RNA were used to generate first-strand cDNA, which was then amplified via the polymerase chain reaction (PCR). In this way, seven overlapping segments of insulin-receptor cDNA were obtained. With the exception of the alternatively spliced 36-base pair exon 11, which is not expressed in lymphoblasts, the complete coding region of the mature proreceptor was examined with a combination of direct sequencing and sequencing of subcloned PCR segments. The nucleotide sequence in both subjects was identical to previously published insulin-receptor cDNA sequences obtained from healthy subjects. These data indicate that abnormalities of insulin binding and receptor function that have been previously observed in vitro with fresh and cultured cells from Pima Indians may be consequences of the diabetic milieu and/or genetic abnormalities in molecules that interact with the insulin receptor.     

Linkage studies on NIDDM and the insulin and insulin-receptor genes

Twenty Black families in which at least two siblings had non-insulin-dependent diabetes mellitus (NIDDM) were typed for restriction-fragment-length polymorphisms at the insulin (INS), insulin-receptor (INSR), and HLA-DR beta loci. Evidence for linkage between NIDDM and these loci was assessed with various genetic models for the transmission of NIDDM and with the affected-sib-pair approach, which does not require assumptions concerning a genetic model for NIDDM. Tight linkage between NIDDM and any of the loci was unlikely under all of the genetic models examined. Similarly, for all three of the loci, the distribution of affected sib pairs sharing 2, 1, or 0 genes identical by descent was not significantly different from (and was very similar to) that expected if the locus were unrelated to disease susceptibility. There was no evidence for linkage heterogeneity for any of the loci when families were grouped according to obesity or age at onset or when considering families individually. We conclude that the INS and INSR loci can be ruled out as major susceptibility loci for NIDDM in most Black families segregating this disorder, but we recognize that defects at either of these loci may cause or contribute to NIDDM in some patients. In addition, it is possible that variation at the INS and/or INSR loci may contribute to NIDDM susceptibility by modifying susceptibility due primarily to another major gene(s) or as part of an overall polygenic component to NIDDM.     

Rat insulin-receptor kinase activity correlates with in vivo insulin action

Tyrosine kinase activity of skeletal muscle-derived insulin receptors isolated from rats that had undergone euglycemic clamps at various insulin infusion rates was examined. Receptors were isolated under conditions designed to preserve their in vivo phosphorylation state, and their kinase activity toward histone was measured in the absence of in vitro exposure to insulin. Results showed that significant activation of the insulin-receptor kinase occurred after exposure in vivo to mean serum insulin concentrations as low as 34 +/- 3.5 microU/ml and that maximal activation was achieved by insulin levels less than or equal to 2000 microU/ml. There was a highly significant correlation between receptor kinase activity and serum insulin concentration in the physiologic range (r = .92, P less than .0001) and between kinase activity and glucose utilization rate (r = .74, P less than .0001). These findings further support a role for the insulin-receptor kinase in insulin action in vivo, and this model provides a novel method for the study of the effect of factors known to influence insulin action on the insulin-receptor kinase under physiologic conditions.     

Skeletal muscle insulin-receptor kinase. Effects of substrate inhibition and diabetes.

Insulin receptor tyrosine kinase activity solubilized from hind limb muscle of control and streptozocin-induced diabetic (STZ-D) rats (2-3 wk) was studied with the substrates histone H2B and poly glutamic acid-tyrosine (glu-tyr) (4:1). Basal and insulin-stimulated kinase activities were inhibited when high concentrations of either substrate were added before initiation of phosphorylation with ATP. Under these conditions, insulin-stimulated activities of diabetic- and control-derived receptor kinase toward H2B were similar at 0.008 mg/ml H2B. However, higher concentrations of H2B (0.04-1 mg/ml) progressively reduced the ratios of diabetic-derived to control-derived receptor kinase activities to approximately 0.5. When inhibition of receptor kinase activities was prevented by allowing maximal autophosphorylation of insulin receptors before addition of H2B, kinase activity of diabetic- and control-derived receptors was similar at all H2B concentrations. Diabetic-derived insulin-receptor tyrosine kinase activity toward poly glu-tyr (4:1) was not significantly different from that of control rats. Under conditions of substrate inhibition (0.4 mg/ml H2B), insulin receptor H2B kinase activity from muscles of rats with severe diabetes (85 mg/kg STZ, 7 days) was significantly decreased, whereas the same activity from rats with moderate diabetes (50 mg/kg STZ, 7 days) was not significantly different from control rats. Insulin receptor alpha,beta dimers were not detectable in muscle preparations from control or diabetic rats. The data suggest that the impairment of muscle-derived insulin-receptor tyrosine kinase activity associated with insulinopenic diabetes reflects, in part, enhanced inhibition by some substrates. If solubilized insulin receptors and the exogenous substrates studied model in vivo events, impaired signaling of the muscle insulin receptor in insulinopenic diabetes may depend on the type and concentration of intracellular tyrosine kinase substrates and the severity of the metabolic derangements.     

Insulin and insulin-receptor autoantibodies in children with newly diagnosed IDDM before insulin therapy

Twenty-nine children, aged 1-15 yr, with newly diagnosed insulin-dependent diabetes mellitus (IDDM) had sera taken before insulin therapy to be examined for the presence of insulin-receptor antibodies by measuring the inhibition of binding of radiolabeled insulin to IM-9 lymphocytes in both whole serum and purified IgG fractions. Groups of children with long-standing IDDM and autoimmune endocrine disease as well as a normal control group were studied. A positive result, defined as binding greater than or equal to 2 SD below the mean zero standard, was found in 3 (10.3%) of the 29 newly diagnosed diabetic patients. As a group, they showed significantly greater binding inhibition than the normal control group for both whole serum and purified IgG (one-tailed t test, P less than .05 and P less than .002, respectively). Insulin autoantibodies were also measured by a sensitive radioimmunoassay technique. A positive result, defined as binding greater than 3 SD above the normal control pooled sera, was found in 9 (37.5%) of 24 of the newly diagnosed IDDM group tested. All 3 subjects positive for insulin-receptor antibodies were also positive for insulin autoantibodies, whereas 6 of the 21 receptor-antibody-negative subjects were positive for insulin autoantibodies (Fisher's exact test, P = .0415). This suggests the possibility that the presence of insulin autoantibodies is a prerequisite for the development of insulin-receptor antibodies, i.e., as an anti-idiotypic response. Insulin-receptor antibodies and insulin autoantibodies may play a currently undefined pathophysiologic role in the development of IDDM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antidiabetic action of vanadyl in rats independent of in vivo insulin-receptor kinase activity

The effects of oral vanadyl sulfate administration for 9-12 days on carbohydrate and lipid metabolism in the basal state and on glucose dynamics during submaximal hyperinsulinemic clamps were investigated in nondiabetic and streptozocin-induced diabetic rats. Decreases in growth rate and water and food consumption were the only significant alterations noted in control animals receiving vanadyl. Administration of vanadyl to diabetic rats resulted in weight loss; a significant decrease in plasma glucose, triglyceride, and cholesterol levels; and decreases in food and water intake, without a concomitant change in plasma insulin concentrations. Vanadyl treatment did not modify either peripheral glucose utilization or hepatic glucose production in control rats during submaximal insulin clamps. In contrast, vanadyl therapy increased insulin-induced glucose utilization significantly and had a small but nonsignificant effect on insulin-mediated suppression of glucose production in diabetic rats. The tyrosine kinase activity of liver- and muscle-derived insulin receptors from diabetic rats that underwent clamp study, which reflected the in vivo phosphorylation state of insulin receptor, was not altered by vanadyl treatment. In conclusion, these results show that augmentation of peripheral glucose utilization is the major determinant of the antidiabetic action of vanadyl and support the notion that the action of vanadyl is independent of insulin-receptor kinase activity.     

Effect of streptozocin-induced diabetes on insulin-receptor tyrosine kinase activity in obese Zucker rats

We examined insulin binding, insulin-stimulated autophosphorylation, and phosphorylation of poly(Glu.Na,Tyr)4:1 by liver and skeletal muscle insulin receptor from lean, obese, and obese streptozocin-induced diabetic Zucker rats. Induction of diabetes with streptozocin (30 mg/kg) lowered the lasting insulin level from 11.4 to 3.8 ng/ml, which was not significantly greater than the lean control level. Autophosphorylation and tyrosine kinase activity of liver insulin receptors were increased 70-100% in the obese control group (relative to lean rats), but diabetes reversed this hyperresponsiveness to insulin. In muscle, obesity was associated with a 40-50% decrease in autophosphorylation and tyrosine kinase activity, which was also reversed in the diabetic state. Autophosphorylation and tyrosine kinase activity were significantly correlated in liver and muscle and were also correlated with fasting insulin levels. These data suggest that insulin-receptor tyrosine kinase activity is regulated differently in liver and muscle and that the abnormalities in kinase activity associated with the obese Zucker rat are at least partly secondary to hyperinsulinemia.     

Evidence for two independent pathways of insulin-receptor internalization in hepatocytes and hepatoma cells

A study of insulin-receptor internalization and recycling was undertaken in primary cultures of rat hepatocytes and a human hepatoma cell line (HepG2). Receptors were quantitated by measuring 125I-insulin binding to partially purified soluble receptor preparations from untreated cells (total receptors) and trypsinized cells (intracellular receptors). In resting HepG2 cells, exposure to insulin results in internalization of insulin receptors, the rate and extent of which is dependent on the insulin concentration. However, receptors do not accumulate inside the cell in proportion to the higher rates of internalization at high concentrations of insulin. This lack of accumulation is explained by much higher recycling rates after exposure to high concentrations of insulin. Similar results were noted for primary cultures of rat hepatocytes. These results imply qualitatively different fates for receptors internalized after exposure to different concentrations of insulin. To further investigate the possibility of different pathways for insulin-receptor internalization and processing, cells in low (1 ng/ml) or high (100 ng/ml) concentrations of insulin were exposed to drugs or treatments known to affect receptor metabolism. Hypotonic shock and hypokalemia, which arrest coated-pit formation, blocked internalization of insulin and insulin receptors at low concentrations of insulin but allowed internalization in response to high concentrations of insulin. The lysosomotropic drugs monensin and chloroquine caused intracellular accumulation of insulin and its receptors internalized at low concentrations of insulin but had a relatively smaller effect on receptors internalized at high concentrations of insulin. All internalization is blocked by 2,4-dinitrophenol. We conclude that high doses of insulin lead to insulin-receptor internalization and recycling through a pathway that is functionally distinct from the pathway taken by receptors internalized by low (physiologic) concentrations of insulin. The pharmacologic experiments raise the possibility that the high-dose pathway, unlike the low-dose pathway, may proceed independently of coated pits and endosomal acidification.