Mutant Insulin Research Articles

Conformational basis of the receptor-binding potency of normal and mutant insulin molecules with relevance to the pathophysiology of noninsulin dependent diabetes mellitus (NIDDM)

Residues 23–26 (Gly-, Phe-, Phe-, Tyr) of the B-chain of insulin constitute a critical area of the receptor-binding region of the molecule. Three chemically distinct mutant insulins have recently been identified in patients with NIDDM, each involving substitution of either B24 of B25 phenylalanine. Two of the mutations have been unambiguously characterized: a B25 phenylalanine-to-leucine substitution [B25(Phe Leu)], and the other, a B24 phenylalanine-to-serine [B24 (Phe Ser)]. We have calculated the preferred conformations of normal and mutant insulins using a global optimization method developed by us earlier. The mutant insulins exhibit significant alterations in conformation and in the average distances between amino acid side-chains as compared to normal insulin. Therefore, the decreased binding affinity of mutant insulin could be either due to an alteration in the nature of the substituted residue (hydrophilic in place of hydrophobic) or to the alteration in one or more critical side-chain distances in the case of a hydrophobic to hydrophobic substitution.

A mutant insulin receptor with defective tyrosine kinase displays no biologic activity and does not undergo endocytosis.

The cDNAs encoding the normal human insulin receptor (HIRc) and a receptor that had lysine residue 1018 replaced by alanine (A/K1018) were used to transfect Rat 1 fibroblasts. Lysine 1018 is a critical residue in the ATP binding site of the tyrosine kinase domain in the receptor beta-subunit. Untransfected Rat 1 cells express 1700 endogenous insulin receptors. Expressed HIRc receptors had levels of insulin-stimulable autophosphorylation in vitro comparable to normal receptors, whereas A/K1018 receptors had less than 1% of that activity. Stimulation by insulin of HIRc receptors in situ in intact cells led to phosphorylation of beta-subunit tyrosine residues and activation of tyrosine kinase activity that could be preserved and assayed in vitro after receptor purification. In contrast, A/K1018 receptors showed no such activation, either of autophosphorylation or of kinase activity toward histone. Cells expressing HIRc receptors display enhanced sensitivity to insulin of 2-deoxyglucose transport and glycogen synthase activity. This increased sensitivity was proportional to insulin receptor number at low but not at high levels of receptor expression. A/K1018 receptors were unable to mediate these biologic effects and actually inhibited insulin's ability to stimulate glucose transport and glycogen synthase through the endogenous Rat 1 receptors. Expressed HIRc receptors mediated insulin internalization and degradation, whereas A/K1018 receptors mediated little, if any. Endocytotic uptake of the expressed A/K1018 insulin receptors was also markedly depressed compared to normal receptors. Unlike HIRc receptors, A/K1018 receptors also fail to undergo down-regulation after long (24 h) exposures to high (170 nM) concentrations of insulin. We conclude the following. 1) Normal human insulin receptors expressed in Rat 1 fibroblasts display active tyrosine-specific kinase, normal intracellular itinerary after endocytosis, and normal coupling to insulin's biologic effects. 2) A receptor mutated to alter the ATP binding site in the tyrosine kinase domain had little if any tyrosine kinase activity. 3) This loss of kinase activity was accompanied by a nearly complete lack of both endocytosis and biologic activity.

The protein-tyrosine kinase activity of the insulin receptor is necessary for insulin-mediated receptor down-regulation.

We have previously demonstrated that the human insulin receptor, mutated in the ATP-binding domain of the beta-subunit, is kinase-defective and fails to mediate multiple post-receptor actions of insulin in stably transfected Chinese hamster ovary cells (Chou, C.-K., Dull, T. J., Russell, D. S., Gherzi, R., Lebwohl, D., Ullrich, A., and Rosen, O. M. (1987) J. Biol. Chem. 262, 1842-1847). This study addresses the role of protein-tyrosine kinase activity in insulin-mediated receptor down-regulation. Although the mutant insulin proreceptor was properly processed and able to bind insulin like the wild-type human receptor, it differed from the latter in the following respects: 1) it failed to mediate internalization of surface-bound radiolabeled ligand; 2) it did not undergo short- or long-term down-regulation in response to 1 microM insulin; 3) it did not exhibit ligand-promoted receptor turnover; and 4) it was not phosphorylated on either tyrosine or serine residues in response to insulin. Although the cells transfected with the mutant receptor failed to respond to insulin-mediated insulin receptor down-regulation, they were able to down-regulate their insulin-like growth factor I receptors in response to insulin-like growth factor I or high concentrations of insulin and were sensitive to monoclonal antibody-induced down-regulation of their insulin receptors. Antibody-mediated receptor internalization alone, however, was unable to mimic at least one action of insulin, thymidine incorporation into DNA, and did not lead to any phosphorylation of the receptor. It is concluded that either the protein-tyrosine kinase activity of the insulin receptor or its phosphorylation state is essential for ligand-mediated receptor down-regulation.

Structurally abnormal insulin in a diabetic patient. Characterization of the mutant insulin A3 (Val----Leu) isolated from the pancreas.

We have recently identified a diabetic patient with marked fasting hyperinsulinemia. Family study revealed that the abnormality was an autosomal dominant trait. High-performance liquid chromatography (HPLC) profile of the patient's serum insulin showed that she had an abnormal insulin in addition to a normal insulin. We have purified her insulin(s) from the specimen of her pancreas, which was biopsied during an operation of cholelithiasis. Insulin was also immunologically purified from the serum of her portal vein. The reverse-phase HPLC analysis revealed that the ratios of normal to abnormal insulin in the pancreas, portal vein, and peripheral vein were 5:4, 4:5, and 1:7, respectively. Radioreceptor assay for insulin using guinea pig kidney membrane revealed that the binding activities of the normal component insulin, the abnormal component insulin and her pancreatic insulin containing both components were 100, 5, and 50% of standard human insulin, respectively. The biological activities of the normal component, the abnormal component and her pancreatic insulin to stimulate glucose oxidation in rat adipocytes were found to be 100, 8, and 60% of standard human insulin, respectively. Analysis of amino acid sequences of the abnormal insulin purified from her pancreas strongly suggested the substitution of leucine for valine at the third position of the A chain, A3 (Val----Leu).

Metabolism of a mutant insulin by a receptor-mediated process and an insulin degrading enzyme

The metabolism of a mutant insulin, [LeuB25]-insulin, was studied in vitro and in vivo. Porcine or mutant insulin (4 micrograms/kg body weight) was injected i.v. into streptozotocin-induced diabetic rats and their plasma glucose and insulin levels were determined. The half-lives of porcine and mutant insulin were 3 min and 18 min, respectively. The ability of the mutant insulin to lower the blood glucose levels was 38% of that of normal when the glucose levels at the nadir were compared. Receptor-mediated degradation of the mutant insulin assessed by chromatography of the degraded materials in the media after incubation with cells was less compared with that of porcine insulin (4% vs. 24%). The media containing the insulin degrading enzyme (IDE) of IM-9 cells and rat livers degraded porcine and mutant insulin to the same extent. These results suggest that the decreased clearance of insulin is due to the decreased receptor binding and the decreased receptor-mediated degradation, but is not due to the decreased degradation by IDE.

Receptor binding and negative cooperativity of a mutant insulin, [Leu A3]-insulin

[Leu A3]-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(>10 −6M). 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.

Prolonged disappearance rate of a structurally abnormal mutant insulin from the circulation in humans.

The first mutant insulin, [LeuB25]insulin, was semisynthesized, and its disappearance rate from the circulation was measured. Three micrograms of normal human or [LeuB25] insulin per kg were administered to three normal subjects. The half-lives of normal insulin and the mutant insulin were 4.5 and 24 min, respectively. The decrement in blood glucose levels after injection of mutant insulin was 22.6% that after injection of normal insulin. The blood glucose-lowering effect of the mutant insulin, evaluated by the time required to reach the nadir, was slightly prolonged (30 vs. 45 min). These results indicate that hyperinsulinemia in patients with abnormal insulin is due to prolonged disappearance because of decreased receptor binding.

Decreased biologic activity and degradation of human [SerB24]-insulin, a second mutant insulin

Decreased biologic activity and degradation of human [SerB24]-insulin, a second mutant insulin

Decreased biologic activity and degradation of human [SerB24]-insulin, a second mutant insulin.

A second mutant insulin, identified as [SerB24]-insulin, has a highly hydrophilic character. To determine the biologic activity and the degradation of this mutant insulin, human [SerB24]- and [SerB25]-insulin analogues were semisynthesized from porcine insulin by an enzyme-assisted coupling method. All of the following studies on isolated rat adipocytes were performed at 37 degrees C to directly correlate the binding potency and the biologic activity. The ability of these insulins to displace 125I-porcine insulin bound to adipocytes was 0.5-2% and 1-4%, respectively, of porcine insulin. When the ability of these insulins to stimulate glucose transport and glucose oxidation was measured, both analogues had full activity at high concentrations (250 ng/ml). However, ED50 of the porcine, [SerB24]-, and [SerB25]-insulins to stimulate glucose transport was 0.37 +/- 0.05, 46.3 +/- 5.4, and 23.3 +/- 5.5 ng/ml, respectively. Similarly, for glucose oxidation, ED50 was 0.38 +/- 0.06, 33.8 +/- 3.6, and 16.6 +/- 3.4 ng/ml, respectively. Thus, the biologic activity of [SerB24]- and [SerB25]-insulins was reduced to 0.5-2% and 1-4% of that of porcine insulin, which was compatible with our previous studies under different conditions. No antagonistic effects were observed for either analogue. Degradation of 125I-labeled [SerB24]- and [SerB25]-insulins was also decreased to 62.8% and 55.8%, respectively, of 125I-porcine insulin. These results confirm the importance of the hydrophobic residues at B24 and B25 in the biologic activity of insulin; the patient having this hydrophilic insulin was considered to be in an insulinopenic state despite the hyperinsulinemia due to decreased degradation of the mutant insulin.

Receptor binding and biological activity of [Ser B24]-insulin, an abnormal mutant insulin

[Ser B24]-insulin, the second structurally abnormal mutant insulin, and [Ser B25]-insulin were semisynthesized and were studied for receptor binding and biological activity. Receptor binding and biological activity determined by its ability to increase 2-deoxy-glucose uptake in rat adipocytes were 0.7–3% of native insulin for [Ser B24]-insulin and 3–8% for [Ser B25]-insulin. Negative cooperative effect of these analogues was also markedly decreased. Immunoreactivity of [Ser B24]-insulin was decreased whereas that of [Ser B25]-insulin was normal. Markedly decreased receptor binding of [Ser B24]-insulin appeared to be due to substitution of hydrophobic amino acid, Phe, with a polar amino acid, Ser, at B24.

Identification of a mutant human insulin predicted to contain a serine-for-phenylalanine substitution.

Using information gained from (i) the relative HPLC retention of an abnormal insulin present in the serum of a hyperinsulinemic diabetic patient and (ii) the loss of an Mbo II restriction site in one of the patient's insulin gene alleles, it was recently predicted that the mutant insulin contained a serine-for-phenylalanine substitution at position B24 or B25. We have now prepared human [SerB24]insulin and [SerB25]insulin by solid-phase peptide synthesis and semisynthesis using an improved approach whereby the protecting groups can be removed from the final product in a single step. During reversed-phase HPLC analysis, the two semisynthetic insulins were clearly separated from normal insulin and from each other. Analysis of the patient's immunoaffinity-purified serum insulin by HPLC and radioimmunoassay showed that the insulin eluted at the position of [SerB24]insulin and coeluted with the analog when the two were studied in admixture. Additional studies showed that [SerB24]insulin and [SerB25]insulin have about 16% and 0.5% of the activity of normal insulin, respectively, in stimulating glucose oxidation by isolated rat adipocytes. We conclude that the patient's abnormal insulin (insulin Los Angeles) is human [SerB24]insulin and that abnormal insulins with amino acid replacements at both positions B24 and B25 can be associated with human diabetes.

LeuB24]insulin is an insulin agonist at the liver in vivo.

A mutant insulin isolated from the plasma of a diabetic patient has been reported to antagonize insulin action in vitro and was thought to be [LeuB24]insulin. This study examines the ability of [LeuB24]insulin to antagonize insulin action at the liver in vivo in anesthetized dogs. Antagonism of insulin action was first simulated by decreasing the intraportal insulin infusion 50%. This resulted in a significant increase in both glucose production (Ra) (delta = + 0.30 +/- 0.08 mg X kg-1 X min-1) and the glucose level in arterial plasma (delta = +6.5 +/- 2.8 mg/dl), validating the responsiveness of the preparation to partial insulin antagonism. [LeuB24]insulin was infused intraportally, at molar ratios of 1:1, 1:2, 1:4, and 1:10 (50, 25, 12.5, and 5 ng/min, respectively) with insulin (54 ng/min). Infusion at all but the lowest dose resulted in a significant drop in glucose production (delta = -0.44 +/- 0.07, -0.35 +/- 0.06, and -0.28 +/- 0.08 mg X kg-1 X min-1 for4 analogue infusions of 50, 25, and 12.5 ng/min, respectively) and plasma glucose levels (delta = -7 +/- 3 and -3 +/- 1 mg/dl for analogue infusions of 50 and 25 ng/min, respectively). No change in Rd (glucose disposal) was observed for either insulin withdrawal of [LeuB24]insulin infusion. We conclude that, at the liver in vivo, [LeuB24]insulin does not antagonize insulin action but rather acts as an insulin agonist. Its hepatic effects would not contribute to a diabetic hyperglycemia.

Identification of a point mutation in the human insulin gene giving rise to a structurally abnormal insulin (insulin Chicago).

Both insulin gene alleles of a diabetic patient with a mutant insulin were cloned in a lambda vector and their nucleotide sequences were determined. Nucleotide sequence analysis revealed, in one allele, a C (cytidylate) to G (guanylate) transversion in the codon for phenylalanine at position 25 of the insulin B-chain. This point mutation leads to the substitution of a leucine for phenylalanine accompanied by the loss of a restriction endonuclease Mboll recognition site and the creation of a new Rsal cleavage site at this position.

Three mutant insulins in man

We have previously identified a structurally abnormal insulin in the serum and pancreas of a middle-aged man with diabetes mellitus which arose from a leucine for phenylalanine substitution at position 24 or 25 of the insulin B chain; further analysis of the patient's leukocyte DNA showed that one of the patient's insulin alleles had undergone mutation resulting in loss of an MboII restriction site normally present in the human insulin gene. Two additional and unrelated patients with the same clinical syndrome have now been identified (ref. 4 and unpublished results). All of these patients showed hyperglycaemia typical of diabetes and with marked hyperinsulinaemia typical of insulin resistance, but all three show normal tolerance to exogenously administered insulin. As the opportunity of examining pancreatic tissue from patients suspected of secreting insulin variants is rare, we have developed a method combining HPLC and radioimmunoassay to identify insulin variants isolated from human sera. By this method we have shown that all three patients noted above secrete structurally variant and chemically distinct insulins. In correction of our original assignment, one is identified as [LeuB25]insulin.

Loss of a restriction endonuclease cleavage site in the gene of a structurally abnormal human insulin

Genomic DNA was isolated from leukocytes of a diabetic patient with a mutant insulin and digested with the restriction endonuclease MboII. Subsequent electrophoresis and hybridization with cloned human insulin cDNA probes revealed the loss of one MboII site consistent with the postulated replacement of a phenylalanine residue at position 24 of the insulin B chain by leucine.

The insulin gene is located on chromosome 11 in humans.

The polypeptide hormone insulin is required for normal glucose homeostasis. Insulin deprivation results in diabetes, a disease affecting up to 5% of the human population1,2. In some animals there are two insulin genes3; however, in most others, including humans, a single gene is present. The conclusion that there is a single insulin gene in humans is based on the finding of a single insulin species and on genetic studies involving mutant insulin alleles4,5. As the pattern of inheritance of insulin defects is not sex linked, the insulin gene must be located on an autosomal chromosome5. Recently, genomic DNA segments containing the human and rat insulin genes have been cloned and the DNA has been sequenced6–8. The fact that mouse and rat insulins are identical3 suggests that the insulin gene sequences and organization are similar. A knowledge of the restriction endonuclease map for the human and mouse insulin genes enables human or mouse insulin DNA sequences to be distinguished in somatic cell hybrids between the two species. In the present study, somatic cell hybrid gene mapping methodologies9 have been used to determine the chromosome localization10 of the human insulin gene. Human–mouse cell hybrids with different numbers and combinations of human chromosomes were examined for the presence of specific human chromosomes and the human insulin gene using cloned human and rat cDNA probes. A 14-kilobase DNA fragment containing the human insulin gene was present in some cell hybrids and was easily distinguished from the DNA fragments containing the mouse insulin gene found in all cell hybrids. Co-existence of the 14-kilobase fragment and chromosome 11 indicate that the insulin gene resolves on chromosome 11 in humans.