Four mutant alleles of the insulin receptor gene associated with genetic syndromes of extreme insulin resistance.

We identified four novel mutant alleles of the insulin receptor gene in three patients with genetic syndromes associated with insulin resistance. Two mutant alleles of the insulin receptor gene were identified in a patient with the Rabson-Mendenhall syndrome who was a compound heterozygote for a mutation at the 3'-splice acceptor site of intron 4 (AG-->GG), the first mutation causing an aberrant splicing at this locus, and a deletion of eight base pairs in exon 12. The second patient with leprechaunism was also a compound heterozygote for a deletion of one base pair in exon 19 and a mutation, Thr910-->Met, which causes impaired receptor processing. Interestingly, the third patient with type A syndrome was a simple heterozygote for the identical one base pair deletion. The fact that the same one base pair deletion links to type A in a simple heterozygote and to leprechaunism in a compound heterozygote appears consistent with the hypothesis that the severity of mutations will determine the phenotype.

A homozygous kinase-defective mutation in the insulin receptor gene in a patient with leprechaunism.

We report a homozygous missense mutation at position 1092 (substitution of glutamine for arginine) in the tyrosine kinase domain of the insulin receptor in a patient with leprechaunism associated with severe insulin resistance and intrauterine growth retardation. Site-directed mutagenesis as well as analyses of the patient's lymphocytes revealed that this mutation causes a marked decrease in tyrosine kinase activity of the insulin receptor without any defect in insulin binding, which causes severe defects in insulin-stimulated glucose transport, glycogen synthesis and DNA synthesis. Thus, this is the first homozygous mutation resulting in a selective-kinase defect of the insulin receptor. Interestingly, the parents who are cousins and are heterozygous for the mutation have type A insulin resistance syndrome. This correlation between genotype and phenotype in a single pedigree suggests that the severity of the mutation will determine the phenotype. Based upon this assumption, we have been successful in prenatal diagnosis of the fifth child. Furthermore, we have demonstrated the effectiveness of clinical administration of insulin-like growth factor-I (IGF-I) in this patient and in vitro analysis of the patient's skin fibroblasts, suggesting that IGF-I can compensate for insulin action via the IGF-I receptor in a patient almost lacking functional insulin receptors.

Signal transduction mechanism of insulin and insulin-like growth factor-1.

Insulin and insulin-like growth factor-1 (IGF-1) are two structurally related hormones which produce similar biological activities such as metabolic and growth promoting actions. Their receptors, insulin and IGF-1 receptors, also share similarities in both structure and functions such as tyrosine-specific protein kinase. We identified insulin receptor substrate-1 (IRS-1) as a common substrate for insulin and IGF-1 receptor tyrosine kinases. We generated IRS-1 knockout mice and showed that IRS-1 plays a physiological role in signal transduction and biological actions of insulin and IGF-1. We also identified pp190 (IRS-2) as an alternative substrate for IRS-1.

A 3-basepair in-frame deletion (delta Leu999) in exon 17 of the insulin receptor gene in a family with insulin resistance.

We studied a woman with acanthosis nigricans and insulin resistance. The patient's Epstein-Barr virus-transformed lymphocytes revealed slightly decreased insulin binding and markedly decreased insulin-stimulated autophosphorylation of the insulin receptor. The nucleotide sequence analysis of the patient's genomic DNA revealed a 3-basepair in-frame deletion in one allele, resulting in the loss of leucine at position 999 of the insulin receptor (delta Leu999). The messenger ribonucleic acid transcripts from the mutant allele in the patient's lymphocytes were not decreased. Insulin-stimulated autophosphorylation of the insulin receptor from cells expressing delta Leu999 mutant insulin receptor complementary DNA was markedly decreased. The proband, her mother, elder brother, and younger brother, who were heterozygous for this mutation, showed moderate or marked hyperinsulinemia during oral glucose tolerance tests. Although fasting glucose levels were normal and fasting insulin values were preserved in all subjects with the mutation for the 8-yr period of observation, a tendency of progressive increase in postload glucose levels was observed. These results suggest that the delta Leu999 mutation, which reduces tyrosine kinase activity, was responsible for insulin resistance and contributed to postload hyperglycemia.

Site-directed mutagenesis of the juxtamembrane domain of the human insulin receptor.

We have studied the functions of the juxtamembrane domain (941-989) of the human insulin receptor by site-directed mutagenesis. Tyrosine phosphorylation of pp185 was impaired in Chinese hamster ovary cells expressing the receptors with the alteration of Tyr960, but not of Tyr953 or Tyr972, to Phe (CHO-Y960F cells) as compared with cells expressing the normal receptors. In CHO-Y960F cells, tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), the activation of phosphatidylinositol 3-kinase in the anti-phosphotyrosine and anti-IRS-1 immunoprecipitates, the activation of mitogen-activated protein (MAP) kinase, and biological actions were also impaired. In addition, although the deletion of residues 954-965 severely impaired insulin internalization, the deletion of NPXY (957-960), the internalization signal of the low density lipoprotein receptor, did not affect internalization. Moreover, neither the deletions around Tyr953 nor the alterations of the tyrosines (953, 960, or 972) significantly reduced internalization. These data suggest that: 1) Tyr960 is important for the recognition of pp185/IRS-1, the association of phosphatidylinositol 3-kinase with pp185/IRS-1, and the activation of MAP kinase; 2) MAP kinase may lie downstream of pp185/IRS-1 in insulin's signal transduction; and 3) the juxtamembrane domain, but not NPXY or individual tyrosines, is important for insulin internalization.

Normal insulin receptor substrate-1 phosphorylation in autophosphorylation-defective truncated insulin receptor. Evidence that phosphorylation of substrates might be sufficient for certain biological effects evoked by insulin.

A mutant human insulin receptor that lacked the 82 amino acids of the COOH terminus of the beta-subunit (del82) was studied. Both the wild type insulin receptor (HIR) and the mutant receptor were expressed in Chinese hamster ovary (CHO) cells by stable transfection. Autophosphorylation and tyrosine kinase activities toward exogenous substrates of solubilized and partially purified del82 were severely impaired. When CHO cells transfected with del82 (CHO-del82) were stimulated with insulin, autophosphorylation was decreased to a great extent compared with cells expressing HIR (CHO-HIR). Nevertheless, tyrosine phosphorylation of an endogenous substrate, pp185, and insulin receptor substrate-1 (IRS-1) in CHO-del82 was comparable with that in CHO-HIR. Insulin-stimulated activation of phosphatidylinositol 3-kinase activity in CHO-del82 was also equivalent to that in CHO-HIR. Moreover, CHO-del82 exhibited the same insulin sensitivity as CHO-HIR with respect to 2-deoxyglucose uptake and thymidine incorporation into DNA. Insulin-induced internalization in CHO-del82 was decreased by 46% as compared with that in CHO-HIR. These data suggest that: 1) the COOH-terminal domain of the insulin receptor may play an inhibitory role in the phosphorylation of pp185 and IRS-1; and 2) phosphorylation of substrates such as pp185 and IRS-1, rather than autophosphorylation of the receptor per se, correlates better with certain biological effects that were mediated by insulin, suggesting that phosphorylation of the substrates might be sufficient for transducing signals downstream.

Site-directed mutagenesis of GLUT1 in helix 7 residue 282 results in perturbation of exofacial ligand binding.

The structure-function relationship of the HepG2/erythrocyte-type glucose transporter (GLUT1) has been studied by in vitro site-directed mutagenesis. Chinese hamster ovary clones in which glucose transporters were transfected were shown by Western blotting with a GLUT1 anti-COOH-terminal peptide antibody to have expression levels of Gln282----Leu, Asn288----Ile, and Asn317----Ile mutations that were comparable with the wild type. All three mutant GLUT1 clones had high 2-deoxy-D-glucose transport activity compared with a nontransfected clone, suggesting that these residues are not absolutely required for the transport function. We have examined the possibility that the inner and outer portions of the transport pathway are structurally separate by measuring the interaction of the mutant transporters with the inside site-specific ligand cytochalasin B and the outside site-specific ligand 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4 -yloxy)-2- propylamine (ATB-BMPA). All three mutant GLUT1 clones showed high levels of cytochalasin B labeling, and the N288I and N317I mutants showed high levels of ATB-BMPA labeling. In contrast to the transport and cytochalasin B labeling results, the transmembrane helix 7 Gln282----Leu mutant was labeled by ATB-BMPA to a level that was only 5% of the level observed in the wild type. We have confirmed that this mutant was defective in the outer site by comparing the inhibition of wild-type and mutant 2-deoxy-D-glucose transport by the outside site-specific ligand 4,6-O-ethylidene-D-glucose. 4,6-O-Ethylidene-D-glucose inhibited wild-type transport with a Ki of approximately 12 mM, but this was increased to greater than 120 mM in the Gln282----Leu mutant. Thus, of the 3 residues mutated in this study, only glutamine 282 substitution causes a major perturbation in function, and this is a specific and striking reduction in the affinity for the outside site-specific ligands ATB-BMPA and 4,6-O-ethylidene-D-glucose.

Effects of fish and safflower oil feeding on subcellular glucose transporter distributions in rat adipocytes.

Effects of fish oil feeding on glucose transport systems and cell size in rat adipocytes were examined and compared with those of safflower oil or carbohydrate feeding under isoenergy intake conditions. Glucose transport activity was assessed by measuring 3-O-methyl-D-glucose transport. The concentration of erythrocyte type glucose transporter (GLUT-1) and muscle/fat type transporter (GLUT-4) was measured by immunoblotting. The amount of each transporter in intact cells was estimated by the amount of transporter and protein of each membrane fraction and by the recovery of marker enzymes. In cells from safflower-fed rats compared with those from carbohydrate-fed rats, insulin-stimulated glucose transport activity per cell decreased to 51% after a 1-wk feeding, and cell size increase became larger with these effects and continued for at least 4 wk. At 1 wk of feeding, GLUT-1 and GLUT-4 per cell in plasma membrane from insulin-treated cells decreased to 62 and 35%, respectively, with concomitant transporter decreases in the low-density microsome fraction. In cells from high-fish oil-fed rats in which two-thirds of safflower oil was replaced by fish oil, when compared with those from safflower oil-fed rats, insulin-stimulated glucose transport activity increased 1.7-fold after 1 wk of feeding with concomitant cellular GLUT-1 and GLUT-4 increases, but its effect declined thereafter. Parallel with this time course, cell size increase was smaller after 1 wk, but this effect also declined thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced insulin-induced mitogenesis and mitogen-activated protein kinase activities in mutant insulin receptors with substitution of two COOH-terminal tyrosine autophosphorylation sites by phenylalanine.

We have studied the function of a mutant human insulin receptor in which two COOH-terminal autophosphorylation sites (Tyr-1316 and -1322) were replaced by phenylalanine (F/Y COOH-terminal 2 tyrosines (CT2)). In addition, we have also constructed a mutant receptor in which Lys-1018 in the ATP-binding site was changed to arginine (R/K 1018). Both the wild type insulin receptor (HIR) and the mutant receptors were expressed in Chinese hamster ovary (CHO) cells by stable transfection. Autophosphorylation of solubilized and partially purified F/Y CT2 was decreased by approximately 30% compared with the HIR. Tyrosine kinase activities of F/Y CT2 and HIR toward exogenous substrates were almost equal. When CHO cells transfected with F/Y CT2 (CHO-F/Y CT2) were stimulated with insulin, autophosphorylation of the beta-subunit of the insulin receptor and the phosphorylation of an endogenous substrate (pp185) in the intact cell were normal compared with cells expressing HIR (CHO-HIR). CHO-F/Y CT2 exhibited the same insulin sensitivity as CHO-HIR with respect to 2-deoxyglucose uptake. However, the dose-response curve of insulin-stimulated thymidine incorporation in CHO-F/Y CT2 was shifted to the left (approximately 5-7-fold) compared with that in CHO-HIR. There was no significant difference in insulin-like growth factor 1-stimulated thymidine incorporation between CHO-F/Y CT2 and CHO-HIR. Furthermore, the dose-response curve of insulin-stimulated kinase activity toward myelin basic protein in CHO-F/Y CT2 was also shifted to the left (approximately 5-fold) compared with that in CHO-HIR. Kinase assays in myelin basic protein-containing gels revealed that both species of MAP kinases (M(r) 44,000, 42,000) were more sensitive to activation by insulin in CHO-F/Y CT2 than in CHO-HIR. This observation was confirmed in immune complex kinase assays toward microtubule-associated protein 2 (MAP2) using specific antibodies against mitogen-activated protein (MAP) kinase. R/K 1018 mutant insulin receptors showed an absence of insulin-stimulated kinase activity and CHO cells transfected with R/K 1018 (CHO-R/K 1018) failed to enhance 2-deoxyglucose uptake or thymidine incorporation in response to insulin. In addition, R/K 1018 kinase-defective insulin receptors were unable to mediate insulin-stimulated MAP kinase activation. These data suggest that: 1) tyrosine kinase activity of the insulin receptor is required for activation of insulin-stimulated MAP kinases and 2) phosphorylation of COOH-terminal tyrosine residues may play an inhibitory role in mitogenic signaling through regulation of MAP kinases.

Expression of M-CSF receptor encoded by c-fms on smooth muscle cells derived from arteriosclerotic lesion.

Vascular smooth muscle cells in atherosclerotic lesions are phenotypically different from those in the normal arterial wall, and no expression of macrophage colony stimulating factor (M-CSF) receptor encoded by the proto-oncogene c-fms has been demonstrated in normal smooth muscle cells. In the present study, we demonstrated expression of c-fms and high affinity binding of M-CSF in smooth muscle cells isolated from an experimental rabbit model of arteriosclerosis (intimal smooth muscle cells), while no expression of c-fms was shown in medial smooth muscle cells. In the immunocytochemical analysis, both types of smooth muscle cells similarly reacted with an antibody specific to muscle cells (HHF 35) but did not react with an antibody specific to rabbit macrophages (RAM 11). In intimal smooth muscle cells, when cells were incubated with acetylated low density lipoproteins (LDL), the binding of acetylated LDL and foam cell formation were observed. In response to M-CSF, tyrosine-phosphorylation, as analyzed by the detection of anti-phosphotyrosine-reactive proteins, and an increased rate of cell proliferation were observed in intimal smooth muscle cells. These results indicated that intimal smooth muscle cells have the characteristics of monocyte-macrophages such as the expression of c-fms, which may be related to their proliferation and phenotypic conversion into foam cells in atheromatous lesions.

Detection of mutations in the insulin receptor gene in patients with insulin resistance by analysis of single-stranded conformational polymorphisms.

We analyzed single-stranded conformational polymorphisms to screen for mutations and polymorphisms in the insulin receptor gene in subjects with or without insulin resistance. Using this new technique, we demonstrated the existence of mutations in the insulin receptor gene which we had identified previously. In addition, a new mutation was found in exon 20 of the insulin receptor gene in a patient with moderate insulin resistance associated with morbid obesity, acanthosis nigricans, and polycystic ovary syndrome. The patient was heterozygous for a mutation substituting Leu (CTG) for Pro (CCG) at codon 1178. Pro1178 is a part of a characteristic sequence motif (D1150 F1151 G1152---A1177 P1178 E1179) common to many protein kinases. Analysis of single-stranded conformational polymorphisms was also used to estimate the frequency of a polymorphism at codon 1058. The two codons CAC (1058 His) and CAT (1058 His) both had a prevalence of 50% in 30 Japanese subjects. These data demonstrate that analysis of single-stranded conformational polymorphisms is a simple and sensitive screening method for mutations and polymorphisms in the insulin receptor gene in subjects with or without insulin resistance. Identification of a mutation in the insulin receptor gene in a patient with a moderate degree of insulin resistance associated with morbid obesity suggests that insulin receptor mutations may exist in patients with Type 2 (non-insulin-dependent) diabetes mellitus associated with a moderate degree of insulin resistance.

In vitro association of phosphatidylinositol 3-kinase activity with the activated insulin receptor tyrosine kinase.

We previously have shown that insulin treatment of cells greatly increases the activity of phosphatidylinositol (PI) 3-kinase in immunoprecipitates made with an antibody to phosphotyrosine. However, the association of PI 3-kinase activity with the activated insulin receptor is not significant under these conditions. In the present study, we have attempted to reconstitute the association of PI 3-kinase activity with the activated insulin receptor in vitro. PI 3-kinase activity does indeed associate with the autophosphorylated insulin receptor in our in vitro system. The autophosphorylation of the insulin receptor and/or its associated conformational change appear to be necessary for the association of PI 3-kinase activity with the receptor, since kinase negative receptor failed to bind PI 3-kinase activity. After binding, PI 3-kinase or its associated protein seems to be released from the activated receptor after the completion of its tyrosine phosphorylation by the receptor. Tyr960 in the juxtamembrane region of the insulin receptor beta-subunit seems to be involved in the association of PI 3-kinase activity with the receptor, but not C terminus region of the beta-subunit including two tyrosine autophosphorylation sites (Tyr1316 and Tyr1322). The in vitro assay system for the association of PI 3-kinase activity with the insulin receptor can be utilized to study the mechanism of interaction of these molecules and will be an useful method to detect other associated molecules with the insulin receptor.

Epidemiology of blindness and visual impairment in Vanuatu.

A population-based survey of blindness was conducted in Vanuatu. Data were gathered on a sample of 3520 of the approximately 150,000 inhabitants of Vanuatu aged at least 6 years, in order to estimate the prevalence and causes of blindness among the whole population. An overall prevalence of blindness of 4.0 per 1000 was found, 85% of which was due to cataract, an avoidable cause of this disability.

Insulin and 12-O-tetradecanoylphorbol-13-acetate activation of two immunologically distinct myelin basic protein/microtubule-associated protein 2 (MBP/MAP2) kinases via de novo phosphorylation of threonine and tyrosine residues.

Two site-specific antibodies have been prepared by immunizing rabbits with chemically synthesized peptides derived from the partial cDNA-predicted amino acid sequence of extracellular signal-regulated kinase 1 (ERK1), which has been proposed to encode the microtubule-associated protein 2 (MAP2) kinase (Boulton, T. G., Yancopoulos, G. D., Gregory, J. S., Slauer, C., Moomaw, C., Hsu, J., and Cobb, M. H. (1990) Science 249, 64-67). With immunoprecipitation in the presence of sodium dodecyl sulfate (SDS) and Western blotting, an antibody to the peptide containing triple tyrosine residues (alpha Y91) resembling one of the insulin receptor autophosphorylation sites specifically recognized 42- and 44-kDa proteins. On the other hand, an antibody to the peptide corresponding to the COOH terminus portions (alpha C92) of the ERK1 cDNA gene product recognized the 44-kDa protein much more efficiently than the 42-kDa protein. With immunoprecipitation in the absence of SDS, alpha Y91 could barely recognize these two proteins and alpha C92 recognized the 44-kDa protein but failed to recognize the 42-kDa protein. Kinase assays in myelin basic protein (MBP)-containing gel, after SDS-polyacrylamide gel electrophoresis, revealed that insulin or 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated MBP kinase activity in alpha Y91 immunoprecipitates comigrated at molecular mass 42 and 44 kDa. On the other hand, the stimulated MBP kinase activity in alpha C92 immunoprecipitates comigrated only at molecular mass 44 kDa. Insulin stimulated the MBP kinase activity in gels and phosphorylation of these two proteins by greater than 10-fold with a maximal level at 5 min. Insulin and TPA rapidly stimulate the phosphorylation of the 42- and 44-kDa proteins via de novo threonine and tyrosine phosphorylation. Tryptic phosphopeptide mapping analysis of the 42- and 44-kDa proteins, respectively, revealed a single major phosphopeptide containing phosphothreonine and phosphotyrosine, which was common to both insulin- and TPA-stimulated phosphoproteins. Protein phosphatase 2A treatment of these two phosphoproteins caused a complete loss of kinase activity with selective dephosphorylation of phosphothreonine. These data strongly suggest that these two proteins are highly related to the mitogen-activated protein (MAP) kinase with an apparent molecular mass of 42 kDa (Ray, L. B., and Sturgill, T. W. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3753-3757) and that these two immunologically similar but distinct MBP/MAP2 kinases may represent isozymic forms of MBP/MAP2 kinases. These data also demonstrate that insulin and TPA activate MBP/MAP2 kinase activity by de novo phosphorylation of threonine and tyrosine residues via a very similar pathway.

Phosphorylation state and biological function of a mutant human insulin receptor Val996.

Chinese hamster ovary (CHO) cell transfectants that expressed human insulin receptors whose glycine 996 was substituted by valine were studied. Receptor processing and insulin binding were unaffected by this mutation; however, this mutant insulin receptor had little or no tyrosine kinase activity. Nevertheless, the Val996 mutant exhibited seryl and threonyl phosphorylation in both the basal and insulin-stimulated state in intact cells. This is in contrast to the Lys----Ala1018 tyrosine kinase deficient mutant (Russell, D. S., Gherzi, R., Johnson, E. L., Chou, C-K., and Rosen, O. M. (1987) J. Biol. Chem. 262, 11833-11840). Cells expressing the normal human receptor were 10-fold more sensitive to insulin than the untransfected CHO cells with respect to phosphorylation of a cellular substrate (pp 185) on tyrosyl residues, glucose incorporation into glycogen, thymidine incorporation into DNA, and phosphorylation of ribosomal protein S6. Cells expressing the mutant receptor exhibited the same insulin sensitivity as the untransfected CHO cells. Insulin was rapidly internalized in cells expressing the normal human receptor and the number of receptors expressed on the cell surface was decreased in response to exposure to insulin. However, little insulin was internalized in cells expressing the mutant receptor, and the number of receptors on the cell surface was not significantly diminished in response to exposure to insulin. It is concluded that despite the occurrence of seryl and threonyl phosphorylations, post-receptor effects of insulin described above are not mediated by the tyrosine kinase-deficient receptor, Val996.

Substrates for insulin-receptor kinase.

Several studies suggest that the tyrosine-specific protein kinase activity of the beta-subunit of the insulin receptor is necessary to mediate the biological effects of insulin. This conclusion leads to the hypothesis that the effect of insulin is mediated through the tyrosine phosphorylation of cellular substrates by the insulin-receptor tyrosine kinase. In this review, the experimental evidence regarding insulin-stimulated phosphorylation of proteins both in vitro and in vivo is evaluated. In a cell-free system, tubulin, microtubule-associated protein 2, tau, fodrin, calmodulin-dependent kinase, calmodulin, and lipocortins 1 and 2 were reported to be good substrates for insulin-receptor kinase. However, none were found to be tyrosine phosphorylated in an intact-cell system. In intact-cell systems, proteins of Mr 185,000 (pp185), 120,000 (pp120), 240,000 (pp240), 15,000 (pp15), 60,000 (pp60), and 62,000 (pp62) as well as several others were reported to be tyrosine phosphorylated in an insulin-dependent fashion. However, the function or functional alteration of these proteins induced by insulin-stimulated tyrosine phosphorylation is not clear. Therefore, physiologically relevant substrates for the insulin-receptor kinase have not been established, and more work is necessary to verify the phosphorylation cascade hypothesis of insulin action.

Autoantibodies to the insulin receptor (B-10) can stimulate tyrosine phosphorylation of the beta-subunit of the insulin receptor and a 185,000 molecular weight protein in rat hepatoma cells.

The immunoglobulin G (IgG) fraction obtained from the serum of a patient (B-10) with type B insulin resistance and acanthosis nigricans stimulated both glucose oxidation in rat adipocytes and autophosphorylation of tyrosine residues in the beta-subunit of insulin receptors in H-35 hepatoma cells. Partially purified insulin receptor from H-35 cells, when incubated with B-10 IgG, had increased tyrosine kinase activity for a synthetic peptide sequentially similar to the site of tyrosine phosphorylation in pp60v-arc (the gene product responsible for cellular transformation by the Rous sarcoma virus). In H-35 cells, both B-10 IgG and insulin stimulated tyrosine phosphorylation in an endogenous 185,000 mol wt protein. This phosphoprotein may be similar to the cellular substrate for insulin in hepatoma and other cultured cell lines demonstrated by others. These results suggest that antiinsulin receptor antibodies (B-10) may initiate their insulin-like effects via tyrosine phosphorylation of the insulin receptor, activation of its tyrosine kinase activity, and phosphorylation of a cellular protein substrate of 185,000 mol wt.