Toshiyasu Sasaoka

A mutation (Trp1193→Leu1193) in the tyrosine kinase domain of the insulin receptor associated with type A syndrome of insulin resistance

SummaryWe evaluated a 35-year-old diabetic male patient with type A insulin resistance, showing acanthosis nigricans. Insulin binding to the patients Epstein-Barr-virus transformed lymphocytes was mildly reduced. The maximal insulin-stimulated autophosphorylation of the insulin receptor from the patients transformed lymphocytes was decreased to 45% of that from the control subjects. On examination, the biological activities of insulin and insulin-like growth factor I in the patients cultured fibroblasts, insulin sensitivity of amino isobutyric acid uptake and thymidine incorporation was decreased, but insulin-like growth factor I action was normal. The sequence analysis of amplified genomic DNA revealed that the patient was heterozygous for a mutation substituting Leu for Trp at codon 1193 in exon 20 of the insulin receptor gene. The patients mother and sister were also heterozygous for a mutation in the insulin receptor gene that substituted Leu for Trp1193 in the Β subunit of the receptor. Therefore, the mutation causes insulin resistance in a dominant fashion. They were less hyperglycaemic and more hyperinsulinaemic than the proband after glucose loading. The mother had diabetes mellitus but did not show acanthosis nigricans, while the sister did not have diabetes and showed acanthosis nigricans. These results suggest that this mutation causes defective tyrosine kinase activity of the insulin receptor, which results in insulin resistance. Insulin action and phenotypic appearance may be mediated by different factors.

Insulin resistance by unprocessed insulin proreceptors point mutation at the cleavage site

Failure to cleave the interconnecting site between alpha- and beta-subunit produced insulin proreceptors in the plasma membranes which had markedly low affinity to insulin, leading to extreme insulin resistance in a patient. We performed cDNA sequence analysis of the cleavage site of the insulin proreceptor from the patient. Polymerase chain reaction was used to obtain large amount of cDNA coding for the region including the interconnecting site. A thermostable DNA polymerase, Taq polymerase, successfully produced enough amount of cDNA of the region to be sequenced. The results showed AGG (Arg) to AGT (Ser) point mutation, resulting in the change of interconnecting sequence of the two subunits from -Arg-Lys-Arg-Arg- to -Arg-Lys-Arg-Ser-. These results suggest that the tertial structure change of the cleavage site leads to production of unprocessed insulin proreceptors.

Receptor binding and negative cooperativity of a mutant insulin, [LeuA3]-insulin

[LeuA3]-insulin, the third mutant insulin, was semisynthesized and was studied for receptor binding and negative cooperative effects. Receptor binding and biological effects of the mutant insulin were 0.3-0.5% of normal, the lowest among three mutant insulins. However, negative cooperative effects of the mutant insulin were almost normal at higher concentration (greater than 10(-6) M). Monoclonal anti-insulin antibody binding studies revealed that carboxyterminal region of B chain was relatively unchanged. These results suggest that N-terminal region of A-chain extending to A3 is important for receptor binding and confirm that A3 does not play an important role for negative cooperativity.

Binding specificity and intramolecular signal transmission of uncleaved insulin proreceptor in transformed lymphocytes from a patient with extreme insulin resistance

SummaryAn alteration of an amino acid sequence in the processing site of the insulin proreceptor by a point mutation of the insulin receptor gene produced extreme insulin resistance. We characterized functional properties of the unprocessed insulin receptor in transformed lymphocytes from a patient. Insulin binding to intact cells and to a partially purified insulin receptor preparation was radically decreased to 20% and 18% of the control values, respectively. In competitive insulin binding to intact cells, [LeuA3]-, [LeuB24]-, [SerB24-insulin, and mini-proinsulin ([B(1–29)-Ala-Ala-Lys-A(1–21)]-insulin) had the same relative binding activity in both the patients and the control cells, but proinsulin and IGF-I were markedly less able to displace 125I-insulin in the patients cells. In contrast to the study in intact cells, proinsulin and IGF-I as well as other insulin analogues had the same relative binding activity to bind to the partially lectin-purified insulin receptor preparations from both the patients and the control cells. As regards the signal transduction after receptor binding, insulin-stimulated autophosphorylation of the unprocessed insulin proreceptor occurred proportionally to the amount of decreased insulin binding. With 0.025% trypsin treatment, the abnormal binding characteristics and autophosphorylation were normalized through conversion to functionally normal receptors. In spite of the abnormal processing, self-association of receptors into oligomeric structures was observed in the proreceptor. These results suggest that the unprocessed insulin proreceptor in the plasma membranes has an altered conformation which affects its binding characteristics but not its intramolecular signal transmission.

Receptor binding and biologic activity of biosynthetic human insulin and mini-proinsulin produced by recombinant gene technology

Human insulin and its precursor, mini-proinsulin, made with a new biosynthetic method, were tested for their receptor binding, biologic action, and antibody binding ability. The structure of mini-proinsulin is similar to that of proinsulin with a shortened C-peptide, B(1-29)-Ala-Ala-Lys-A(1-21) insulin. The ability of biosynthetic human insulin to bind to receptors, to stimulate 2-deoxyglucose uptake in isolated adipocytes, and to bind to insulin antibody was comparable to that of semisynthetic human insulin. The ability of mini-proinsulin to bind to insulin receptors and to stimulate 2-deoxyglucose uptake in adipocytes was 0.5 and 0.2% that of human insulin, whereas the corresponding abilities of proinsulin were 5 and 3%, respectively. Despite having less receptor binding and biologic activity, mini-proinsulin demonstrated higher affinity for the insulin antibody than did proinsulin. These results suggest that biosynthetic human insulin behaves similarly to semisynthetic human insulin in its receptor binding and biologic activity, and that the shortened C-peptide region reduces receptor binding by fixing or covering the N-terminal region of the A chain, which is important for receptor binding.

Unprocessed insulin proreceptor in cultured fibroblasts from a patient with extreme insulin resistance

Insulin receptors and IGF-I receptors in cultured fibroblasts were investigated in a patient with extreme insulin resistance due to unprocessed insulin receptors. Insulin binding to cultured fibroblast monolayers and partially purified insulin receptors was extremely decreased to 27% and 18% of control value, respectively. Affinity cross-linking study revealed that molecular weight of the insulin receptor was 210 kDa and that it could not be dissociated to alpha- and beta-subunit with dithiothreitol treatment. Because IGF-I binding to the fibroblasts from the patient was normal and alpha-subunit of IGF-I receptor was 135 KDa, the defect was specific to the insulin receptor. Autophosphorylation of the 210 kDa unprocessed insulin proreceptor was stimulated by insulin in a dose-dependent manner. In the fibroblasts from the patient, insulin-stimulated alpha-aminoisobutyric acid uptake was fivefold shifted to the right in the dose-response curve (ED50 20 ng/mL for the patient v 3.5 ng/mL for the control subjects), but the maximally stimulated uptake was normal. With 0.025% trypsin treatment, insulin binding and alpha-aminoisobutyric acid uptake were normalized. These results suggested that (1) abnormal processing of insulin proreceptor also occurred in the cultured fibroblasts, (2) the postreceptor steps of insulin action were totally intact, and (3) IGF-I receptors were normally processed in this patient.

Transfection of cDNA with G→T point mutation at the cleavage site of insulin receptors to cos 7 cells

To study whether the G----T point mutation of insulin proreceptors at the cleavage site which changed -Arg-Lys-Arg-Arg- to -Arg-Lys-Arg-Ser- caused unprocessed insulin receptors with decreased insulin binding affinity, we performed transfection of cDNA with the mutation in COS 7 cells and examined the expressed insulin receptors. After site-directed mutagenesis, an expression vector pGEM3SV was used to make a plasmid which contained full-length HIRcDNA behind SV40 early promoter. Transfection of normal HIR cDNA produced normal insulin receptors on the plasma membranes in COS 7 cells. However, transfection of cDNA with the mutation resulted in the presence of 210K proreceptors in the plasma membranes with decreased insulin binding ability (35% of normal). These results suggest that the mutation, not the defect of converting enzyme, was the cause for unprocessed insulin proreceptors in the patients with insulin resistance.

W-7 specifically inhibits insulin-induced increase in glucose transport

To elucidate the role of calmodulin in insulin action, we examined the effect of the calmodulin antagonists, W-7 and W-5, on glucose transport in isolated rat adipocytes. W-7 inhibited insulin-stimulated 2-deoxyglucose uptake by 18% at 100 microM, but it did not affect basal uptake levels. W-5, a less potent analogue of W-7, however, had no significant effect at the same concentration, indicating that the effect was specific to calmodulin. Similar results were observed in a 3-O-methylglucose uptake study. Kinetic analysis of 2-deoxyglucose uptake revealed that W-7 affected the insulin-induced increase in Vmax but not Km. These results suggest that calmodulin modifies insulin action in the glucose transport system.

Unprocessed insulin proreceptors due to point mutation at the cleavage site

Two sisters presented with severe insulin resistance and markedly decreased insulin binding to erythrocytes, cultured fibroblasts and transformed lymphocytes. The dose-response curve of insulin-stimulated amino acid uptake in the fibroblasts was shifted to the right. The molecular weight of the insulin receptor on the transformed lymphocytes from the patients was 210,000 and could not be dissociated to alpha- and beta-subunits by dithiothreitol treatment. However, the proreceptor was cleaved by trypsin and this led to the production of alpha-subunit with normal insulin binding. We performed cDNA sequence analysis of the cleavage site of the insulin proreceptor from the patients. The polymerase chain reaction was used to obtain a large amount of cDNA coding for the region including the interconnecting site. A thermostable DNA polymerase, Taq polymerase, successfully produced enough cDNA for the region to be sequenced. The results showed an AGG (Arg) to AGT (Ser) point mutation, resulting in the change of the interconnecting sequence of the two subunits from -Arg-Lys-Arg-Arg- to -Arg-Lys-Arg-Ser-. These results suggest that the tertiary structure change of the cleavage site leads to production of unprocessed insulin proreceptors.