Atypical antiinsulin receptor antibodies in a patient with type B insulin resistance and scleroderma

We studied a 23-yr-old woman with scleroderma and type B insulin resistance. The association with autoimmune disease suggested that the insulin resistance resulted from autoantibodies to the insulin receptor. However, in preliminary studies, serum antireceptor antibodies were not detected in an assay that measures the ability of the antibodies to inhibit insulin binding to the insulin receptor. Antireceptor antibodies were subsequently detected by their ability to immunoprecipitate affinity-labeled receptors. After the patient had received immunosuppressive therapy with prednisone and cyclophosphamide for 3 months, her insulin resistance remitted, and she developed hypoglycemia. Simultaneously with the remission of insulin resistance, the titer of serum antireceptor antibody (measured by the immunoprecipitation assay) fell to less than 1% of the previous level. In a series of 21 patients, this is the first patient with antireceptor antibodies that bound to the insulin receptor without inhibiting insulin binding.     

Monoclonal antibodies to the human insulin receptor block insulin binding and inhibit insulin action.

Antibodies to the insulin receptor were prepared in BALB/c mice by immunization with IM-9 human lymphocytes, a cell type that has a large number of plasma membrane insulin receptors. The spleens of these mice were then removed, and their lymphocytes were fused to a mouse myeloma cell line, FO cells. After screening over 1,200 resulting hybrids, one stable hybrid was obtained that produced IgG1 antibodies directed towards the insulin receptor. This antibody blocked 125I-labeled insulin binding to its receptor by more than 90% in three human tissues: IM-9 cultured lymphocytes, freshly isolated adipocytes, and placenta membranes. In contrast, the antibody did not inhibit insulin binding to rat adipocytes and rat liver plasma membranes, suggesting that the antibody was species specific. In IM-9 cells, which had their proteins prelabeled with [35S]methionine, the antibody precipitated two polypeptides with molecular weights of 135,000 and 95,000; these molecular weights are identical to those previously identified as the alpha and beta subunits of the insulin receptor. The monoclonal antibody inhibited the actions of insulin on both human adipocytes and fibroblasts, suggesting that the antibody was an antagonist of insulin action. The present studies suggest, therefore, that monoclonal antibodies to the insulin receptor may provide new insights into the structure of the insulin receptor and its interaction with insulin.     

胰岛素抵抗机制的探研——阻断型胰岛素抗体是导致胰岛素抵抗的原因之一

目的　探讨胰岛素抗体导致胰岛素抵抗发生的机制。方法　利用单抗制备技术和酶联免疫方法获得１７ 株抗人胰岛素单克隆抗体（ＭＡｂｓ） ,测定这些抗体阻断胰岛素与其受体结合、抑制ＣＨＯ 细胞（Ｃｈｉｎｅｓｅｈａｍｓｔｅｒｏｖａｒｙｃｅｌｌｓ）上胰岛素受体自身磷酸化、以及免疫结合共价交联的胰岛素胰岛素受体的能力。结果　在这组ＭＡｂｓ 中,多数ＭＡｂｓ（１６／１７）阻断胰岛素与其受体结合或抑制ＣＨＯ细胞上胰岛素受体自身磷酸化,其识别胰岛素上的位点与胰岛素的受体结合区域相重叠。有１ 株ＭＡｂ 具有免疫结合已与受体交联的胰岛素的能力,但不阻断胰岛素的作用。结论　在胰岛素抗体阳性的糖尿病患者中,其胰岛素抗体的识别位点对胰岛素抵抗的发生有重大作用。     

Monoclonal antibodies to the insulin receptor mimic metabolic effects of insulin but do not stimulate receptor autophosphorylation in transfected NIH 3T3 fibroblasts.

The metabolic actions of insulin and anti-insulin receptor monoclonal antibodies were compared with their effects on insulin receptor phosphorylation in mouse NIH 3T3 fibroblasts transfected with human insulin receptor cDNA. In serum-starved NIH 3T3 HIR3.5 cells, uptake of 2-deoxy-[3H]glucose was stimulated up to 2-fold after 30 min with insulin, with a half-maximal effect at 0.1 nM insulin. Incorporation of [3H]thymidine was stimulated approximately 12-fold after a 16-hr preincubation with insulin, with a half-maximal effect at 2 nM insulin. Phosphorylation of insulin receptor beta-subunit in cells prelabeled with [32P]phosphate was increased 10- to 20-fold within 5 min of adding insulin, with a half-maximal effect at approximately 3 nM insulin. Monoclonal antibodies reacting with four different epitopes on the insulin receptor mimicked the effect of insulin on 2-deoxyglucose uptake. These antibodies also stimulated thymidine incorporation, although the maximum stimulation was only approximately 30% that of insulin. Two antibodies (25-49 and 83-14) showed a similar concentration dependence to insulin in their metabolic effects and in the inhibition of 125I-labeled insulin binding to cells. The other two antibodies (83-7 and 18-44) were somewhat less potent and did not inhibit insulin binding. None of the antibodies significantly increased insulin receptor phosphorylation at concentrations up to 100 nM, which at least in the case of 25-49 and 83-14 was sufficient for full receptor occupancy. It is concluded that the insulin-like metabolic effects of antibodies involve a mechanism of receptor activation that is independent of autophosphorylation and hence that receptor autophosphorylation is not an essential step in triggering at least some events in the insulin signaling pathway.     

Successful control of a case of severe insulin allergy with liraglutide

A 72‐year‐old woman presented with repeated hypoglycemic and hyperglycemic episodes because of an insulin allergy. On admission, she was diagnosed with type B insulin resistance syndrome. She was also found to have anti‐insulin antibodies. After steroid therapy, glycemic control improved dramatically accompanied by the disappearance of the insulin allergy. We then introduced liraglutide, which successfully stabilized her glycemic episodes without allergic reactions. Liraglutide might be useful to treat patients with severe insulin allergy.     

Clinical efficacy of insulin-like growth factor-1 in a patient with autoantibodies to insulin receptors: a case report

The type B insulin-resistance syndrome is characterized by the presence of anti-insulin receptor antibodies that cause severe insulin resistance. Treatments including steroids, cyclophosphamide, plasmapheresis, or insulin-like growth factor-1 (IGF-1) are chosen according to severity of insulin resistance. We describe a patient with type B insulin resistance syndrome who was treated successfully with human recombinant (hr) IGF-1, although this treatment provoked a severe allergic reaction. An elderly man with impaired glucose tolerance and unpredictable hypoglycemic episodes which were gradually worsening increased in hemoglobin (Hb)A1c concentration from 6.5 to 13.4%. His fasting and postprandial hyperglycemia were associated with severe hyperinsulinemia. The patient was diagnosed with type B insulin-resistance syndrome by the presence of anti-insulin receptor antibodies. Double-filtration plasmapheresis, plasma exchange, and immunosuppressive therapy with cyclophosphamide and cyclosporin all failed to suppress anti-insulin receptor antibodies more than transiently. When we attempted the treatment by daily administration of hrIGF-1, fasting and postprandial plasma glucose concentrations became normal and HbA1c levels decreased to 7.1% over 2 months, until on one occasion administration resulted in anaphylaxis. After the patient became stable, desensitization therapy was performed successfully, and hrIGF-1 could be administered again with the plasma glucose returning. We concluded that IGF-1 therapy was an effective treatment choice for type B insulin-resistance syndrome in cases whose plasma exchange and immunosuppressive therapy have failed.     

Diagnostic algorithm and management of immune-mediated complications associated with subcutaneous insulin therapy.

Immune mediated complications associated with subcutaneous insulin therapy such as insulin neutralizing antibodies and/or skin reactions are rare conditions since human insulin is in general use. Nevertheless, if it occurs, a stepwise diagnostic approach is essential for differential diagnosis and consecutive treatment of these complications. Here we suggest a diagnostic algorithm to deal with e.g. insulin antibody formation of the IgG and/or IgE type and/or severe skin reactions resulting in poor metabolic control and often "brittle diabetes" in affected patients. This diagnostic algorithm includes step 1: Intradermal skin testing with positive and negative controls, additives and different insulin preparations; step 2: Quantification of insulin specific IgG and IgE in the serum, and step 3: Analysis of the time dependent binding/dissociation curves of the insulin neutralizing antibodies in an ex vivo/in vitro assay to assess the clinical significance of these antibodies. Based on 158 insulin treated control subjects and four patients with typical symptoms and signs representing the spectrum of immune-mediated complications subsequent to subcutaneous insulin therapy we demonstrate that the proposed stepwise approach leads to a definite diagnosis as a prerequisite for individual and successful therapy.     

A case of leprechaunism with extreme insulin resistance due to a primary defect in insulin receptors

This report describes a 3-month-old female infant with the typical physical features of leprechaunism. The patient demonstrated glucose intolerance and marked hyperinsulinemia (4600 microU/ml). Since an intravenous insulin injection (actrapid insulin: 0.15 U/kg) caused no significant decrease in the blood glucose level, the presence of insulin resistance was suggested. Neither insulin antibodies nor insulin receptor antibodies were were found in the patient's plasma, and other circulating insulin antagonists such as glucagon, growth hormone, and cortisol were within normal limits. [125I]Insulin binding to the erythrocytes from the patient was as low as 1.02% (control infants: 4.89 +/- 1.08% [mean +/- SD]). [125I]Insulin binding to the cultured transformed lymphocytes from the patient was similarly reduced to 3.58% (control: 20.9 +/- 2.71% [mean +/- SD]). From these findings we concluded that the insulin resistance was due to a primary defect in insulin receptors. Interestingly, transient remissions of the patient's glucose intolerance and hyperinsulinemia were observed during a year of follow-up study. The insulin tolerance test which was performed at the remission period showed an improvement in insulin resistance. However, the insulin binding defect to erythrocytes remained unchanged even at the remission period. The exact cause of these remissions was not clear and remained to be elucidated.     

Monoclonal antibodies to the human insulin receptor that activate glucose transport but not insulin receptor kinase activity.

Three mouse monoclonal antibodies were produced that reacted with the alpha subunit of the human insulin receptor. All three both immunoprecipitated 125I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited 125I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity.     

Hypoglycaemia induced by antibodies to insulin receptor following a bone marrow transplantation in an immunodeficient child

Summary Severe hypoglycaemia developed seven months after a bone marrow transplantation in a child with severe combined immunodeficiency. His serum exerted potent insulin-like activity: (a) it stimulated insulin receptor autophosphorylation and kinase activity in cell-free systems, this effect being additive to insulin; (b) it increased glucose transport in isolated soleus muscle. These insulin-like effects were due to immunoglobulins against the insulin receptor. Indeed, the patient serum immunoprecipitated human or murine insulin receptors from different tissues and inhibited insulin binding to receptor on human IM-9 lymphocytes. After corticoids and immunosuppressive therapy by azathioprine, the patient hypoglycaemic episodes disappeared, and concomitantly, the antibodies to insulin receptor were no longer detected, as judged by both immunoprecipitation of insulin receptor and stimulation of glucose transport.     

Differential modulation of insulin actions by dexamethasone: studies in primary cultures of adult rat hepatocytes

BACKGROUND/AIMS: Steroid diabetes is associated with hepatic insulin resistance; in hepatic cell models, however, mainly insulin-permissive effects have been described. Here we investigate modulation by dexamethasone of a larger number of insulin actions. METHODS: Adult rat hepatocytes were cultured+/-dexamethasone for 48 h; insulin actions were studied subsequently. RESULTS: Stimulation of glycolysis by insulin but not by glucose required culture with dexamethasone. Activation of glycogen synthesis by insulin or glucose was strongly enhanced by dexamethasone, the insulin effects on glycogenolysis and amino acid uptake were not modulated. When dexamethasone was omitted from the culture, insulin was incapable to activate glycogen synthase, inactivate glycogen phosphorylase or elevate the level of fructose 2,6-bisphosphate. Dexamethasone did not alter insulin binding, insulin receptor number or kinase activity, insulin receptor substrate-1 and Akt protein expression/phosphorylation. Insulin-stimulated association of phosphatidylinositol 3-kinase with insulin receptor substrates-1 and -2 was increased with dexamethasone, the increased association with IRS-2 may, at least partially, be explained by higher IRS-2 protein expression. CONCLUSIONS: The steroid does not cause hepatic resistance in vitro. The differential attenuation under steroid deprivation points to defects in branches of the insulin signal chain and/or loss of hormonal regulation at the level of target enzymes.     

Antiinsulin antibodies and clinical characteristics of patients with systemic lupus erythematosus and other connective tissue diseases with steroid induced diabetes

The development of antiinsulin antibodies during insulin therapy for steroid induced diabetes was documented in a group of patients with systemic lupus erythematosus and other connective tissue disorders. No patient had antiinsulin antibodies due to the underlying disorder, but all developed antibodies after treatment with exogenous insulin while receiving steroid therapy. Most antibodies had higher avidity for beef insulin, suggesting that pork or human insulin may be of benefit in the management of these patients.     

Comparison of human and porcine insulin therapies in children with newly diagnosed diabetes mellitus

Summary A multicenter, longitudinal study of children below the age of 16 years with newly diagnosed Type 1 (insulin-dependent) diabetes treated either with porcine monocomponent insulin (n=26) or semisynthetic human monocomponent insulin (n=26) was performed during the first 24 months after onset of diabetes. The two groups were carefully matched for age, duration of disease symptoms, initial metabolic values, islet cell antibodies and HLA-DR antigens. During the 24-month observation period there was no significant difference between the two groups in respect to the clinical course, insulin dosage, HbA₁ and residual B-cell activity. No child in either group had a real remission without necessitating insulin therapy. The prevalence of insulin antibodies increased slowly and was 62% in the group treated by human insulin and 52% in the porcine insulin-treated group after 24 months. The titres were generally low and there was no statistical difference between the two groups in respect to insulin antibody formation.     

The insulin receptor: characterization and regulation using insulin-antiinsulin antibody complexes as a probe for flow cytometry

The primary approach for the characterization of the insulin receptor has been through the study of its interaction with 125I-labeled insulin. Recently, we demonstrated that insulin receptors can also be identified by flow cytometry using antibodies to the receptor. In the present study, we characterized the insulin receptor on human lymphoblastoid cells (IM-9) and studied its regulation using insulin and antiinsulin antibodies as a probe for flow cytometry. The mean peak fluorescence of the cells treated with insulin followed by antiinsulin serum was 30-50 U above the control value. There was a close correlation between [125I]insulin binding and peak fluorescence. Fish insulin, which has about 50% the affinity of porcine insulin for the insulin receptor but does not bind to antiinsulin antibodies, did not enhance antiinsulin antibody binding, but competed for the pork insulin-antiinsulin antibody complexes in a dose-dependent manner. Exposure of IM-9 cells to insulin or antireceptor antibodies resulted in reduction in the number of insulin receptors. Cells down-regulated with 10(-6) M insulin or a monoclonal antibody to the insulin receptor had 40% of the [125I]insulin binding of the control cells and 40-50% of the peak fluorescence when insulin-antiinsulin was the probe for the immunofluorescence studies. Cells down-regulated with human autoantibodies to the receptor had 4% [125I]insulin binding and 10% peak fluorescence. In all cases, receptors were lost proportionally from all cells, yielding a single symmetrical fluorescent peak. These date indicate that flow cytometry with insulin-antiinsulin antibody complexes provides a new approach to the measurement of insulin receptors, since it provides direct measurement of the occupied receptor.     

Enhanced efficacy of U-500 insulin in the treatment of insulin resistance caused by target tissue insensitivity

This report describes a 55-year-old diabetic patient with severe insulin resistance due to obesity and hepatic cirrhosis. Anti-insulin antibodies were responsible for only a minor part of the insulin resistance. Insulin resistance was mediated primarily at the target tissue level by a combination of insulin receptor deficiency and a postreceptor defect. When the patient was treated with U-500 regular pork insulin subcutaneously, her insulin requirement was only one third to one half of that during U-100 NPH or regular insulin treatment. The reasons for the greater efficacy of U-500 insulin are unknown. U-500 insulin may have a place in the optimization of therapy for patients with insulin resistance of nonimmunologic origin.     

Disappearance rate of insulin antibodies after discontinuing insulin treatment in 42 Type 2 (non-insulin-dependent) diabetic patients

Summary The disappearance rate of insulin antibodies was studied after cessation of insulin treatment which had been given for 3 months to 6 years in 42 Type 2 (non-insulin-dependent) diabetic patients. Insulin antibodies were measured before and 15 days after interruption of insulin treatment, and every 30 days until the disappearance of insulin antibodies. The mean ± SD value of insulin binding in the entire group before the interruption of insulin treatment was 32±14%. There was no relationship between the antibody level at that time and the duration of insulin treatment. However, the insulin antibody level was significantly higher in 17 diabetic patients on an insulin dose of >20 U/day (p<0.02) than in 25 on an insulin dose of <20 U/day (39±13% versus 28±12%). A positive correlation was found between intial insulin binding and the time required for it to fall below 10% (r = 0.74). Antibodies were absent 60 days after discontinuing insulin treatment in eight of ten subjects presenting with initial binding of <20%. In contrast, in only two of 12 patients with an initial binding of >40% were insulin antibodies detectable 150 days after discontinuation of insulin therapy. Disappearance of insulin antibodies sometimes took up to 1 year and occasionally even more than 2 years.     

Insulin lispro reduces insulin antibodies in a patient with type 2 diabetes with immunological insulin resistance

We describe a 54-year-old Japanese female with type 2 diabetes admitted to our hospital with poor metabolic control. On admission the patient's HbA1c was 9.1% despite having taken over 60 U of human insulin per day for the previous 10 years. A high titer of antibodies to insulin was detected in the serum, and therefore, we decided to introduce insulin lispro with the aim of minimizing immunogenicity. Over the next 2 months, the dosage of insulin required to achieve reasonable blood glucose control was reduced, with the HbA1c level decreasing significantly to 6.8%. The patient was subsequently withdrawn from insulin and discharged on diet therapy only. This case demonstrates that insulin lispro may ameliorate resistance to insulin therapy even in the presence of human insulin antibodies.     

Characterization of purified autoantibodies to the insulin receptor from six patients with type B insulin resistance.

Anti-insulin-receptor autoantibodies are present in the serum of patients with the type B syndrome of extreme insulin resistance. Sera from six patients with this syndrome were purified over protein-A agarose to remove insulin and other serum factors and obtain an immunoglobulin fraction. These purified fractions were used to quantitatively determine the antibodies' activity in three separate assays. The ability to inhibit insulin binding was determined in an assay using fibroblasts that overexpress the human insulin receptor; the ability to immunoprecipitate the receptor was determined in an assay using biosynthetically labeled insulin receptors rather than insulin cross-linked receptors; and the ability to stimulate glucose oxidation was determined in isolated adipocytes. We show that the ability of these antibodies to inhibit insulin binding is tightly coupled to their ability to immunoprecipitate the biosynthetically labeled receptor, but neither assay predicts the bioactivity of these immunoglobulins. We suggest that the inability to show this tight coupling in the past may be due to methodological differences. We find no evidence that these antibodies are anti-idiotypic antibodies.     

Impact of insulin antibodies on insulin aspart pharmacokinetics and pharmacodynamics after 12-week treatment with multiple daily injections of biphasic insulin aspart 30 in patients with type 1 diabetes

OBJECTIVE: This study aimed to evaluate the impact of insulin antibodies on insulin aspart pharmaco-kinetics and pharmacodynamics after 12-week multiple daily injections of biphasic insulin aspart 30 (30% fast-acting and 70% protamine-crystallised insulin aspart, BIAsp30) in patients with type 1 diabetes. METHODS: Twenty-three patients (8 women, 15 men) aged 44.8 (20.6-62.5) years (median and range) with diabetes duration of 19.5 (1.6-44.6) years and haemoglobin (Hb)A(1C) of 9.2% (8.1-12.3%) participated in the study, which consisted of 12-week treatment with multiple injections of BIAsp30. At the end of the treatment period, all patients attended two 24-h profile days 1 week apart for pharmacokinetic and pharmacodynamic assessments. HbA(1C) and insulin antibodies were also determined. RESULTS: Patients were stratified into two groups depending on whether the level of insulin binding to insulin antibodies was below or above 75% (moderate vs high (%, median and range): 62 (15-74) vs 80 (75-89)). High levels of insulin antibodies resulted in about threefold increase in AUC((0 - 24 h)) (the area under the concentration-time curve during 24 h) for total insulin aspart (analysis of variance, P < 0.05). The differences in free insulin aspart pharmacokinetics, insulin pharmacodynamics and HbA(1C) were not statistically significant between patients with different levels of insulin antibodies. Total daily insulin dosage was significantly lower in patients with high than moderate levels of insulin antibodies. CONCLUSIONS: In type 1 diabetic patients, high levels of circulating insulin antibodies result in elevated total, but not free, insulin aspart profiles. Consistent with the finding of similar insulin pharmacodynamics, the long-term glycaemic control is not significantly different between patients with different levels of insulin antibodies.     

Relationship between insulin A chain regions and insulin biological activities

AIM:To study the relationship between insulin A chain regions and insulin biological activities, we designed a series of insulin analogues with changes at A21, A12-18 of C terminal helical region and A8-10 located in the region of A6-A11 intra-chain disulphide bond.METHODS:Insulin A-chain analogues were prepared by stepwise Fmoc solid phase manual synthesis and then combined with natural B-chain of porcine insulin to yield corresponding insulin analogues. Their biological activities were tested by receptor binding, mouse convulsion and immunological assay.RESULTS: A21Ala Ins retains 70.3% receptor binding capacity and 60% in vivo biological activity.DesA13-14, A21Ala Ins and DesA12-13-14-15, A21Ala Ins still have definite biological activity,7.9% and 4.0% receptor binding,and 6.2% and 3.3% in vivo biological activity respectively. A15Asn, A17Pro, A21Ala Ins maintains 10.4% receptor binding and 10% in vivo biological activity. A8His, A9Arg, A10Pro, A21Ala Ins, A8His, A9Lys, A10Pro, A21Ala Ins and A8His, A9Lys, A10Arg, A21Ala Ins have 51.9%, 44.3% and 32.1% receptor binding respectively,50%, 40% and 30% in vivo biological activity respectively, and 28.8%, 29.6% and 15.4% immunological activity respectively.CONCLUSION:A21Asn can be replaced by simple amino acid residues.The A chains with gradually damaged structural integrity in A12-18 helical region and the demolition of the A12-18 helical region by the substitution of Pro and Asn for A17Glu and A15Gln respectively can combine with the B chain and the combination products show definite biological activity, the helical structure of A12-18 is essential for biological activities of insulin. A8-10 is not much concerned with biological activities, but is much more important antigenically in binding to its antibodies, these results may help us design a new type of insulin analogue molecule.