Insulin antibodies and hypoglycemia in diabetic patients

Metabolism, 1986

To determine the influence of insulin antibodies (and their equilibrium kinetic properties) on the pharmacokinetics of insulin, we examined the relationship between insulin antibody binding and the initial rate of increase, time to peek, and return to baseline of therapeutic doses of insulin injected subcutaneously (0.15 U/kg) and the half-life, distribution space, and metabolic clearance rate of intravenously infused insulin (2 mU/kg/min) in insulin-treated patients with diabetes mellitus. Compared to age-weight-matched nondiabetic subjects, the diabetic subjects had reduced initial rates of increase (0.33 + 0.2 Y 0.44 + 0.03 ~U/mL/min, P < 0.05). delayed time to peak (130 + 12 Y 88 * 8 min. p < 0.02). and prolonged return to baseline (486 + 37 Y 313 + 13 min. P < 0.01 I of plasma free insulin levels after subcutaneous injection of insulin, and a prolonged half-life (18.8 f 5.8 ~4.3 + 0.3 min. P < 0.02). increased distribution space (804 + 284 v 108 + 10 mL/kg, P < 0.001). and augmented metabolic clearance rate (28.5 + 1.8 Y 17.3 + 0.7 mL/kg/min, P < 0.001) after intravenously infused insulin. All of these abnormal parameters were significantly correlated with binding of insulin to insulin antibodies at tracer insulin concentrations (Bo) and with the high affinity of insulin antibody binding sites as determined by Scatchard analysis. However, patients with 'zsl insulin antibody binding (Bo) less than 10 percent had normal or near normal plasma free insulin pharmacokinetics.

Acta Diabetologica, 2008

To evaluate ex vivo/in vitro the binding and dissociation characteristics and the level of crossreactivity of insulin antibodies and insulin autoantibodies directed to three different insulin molecules (human, bovine and porcine insulin). In this study sera from 17 diabetic patients were included, who were exclusively treated with s.c. human insulin, but presenting with severe insulin antibody mediated, immunological insulin resistance (i.e., insulin antibodies, IA). In addition, we included serum from one female patient, previously diagnosed with insulin autoimmune syndrome (no exposure to exogenous insulin treatment, i.e., insulin autoantibodies, IAA). Antibody concentrations and a binding/dissociation analysis was performed by using J 125-labelled (position: A-14) human, porcine and bovine insulin according to the protocol described recently. In the patient with insulin autoimmune syndrome (IAA) we observed total crossreactivity between human, bovine and porcine insulin. By contrast, in the group of s.c. insulin treated diabetic patients with antibodymediated insulin resistance (IA) we detected only partial crossreactivity. In these patients, there was a significantly higher level in the inital insulin binding (P \ 0.05) directed to human insulin (median: 34%, IQR: 21.0-62.0), compared to porcine (median: 29.5%, IQR: 18.3-61.0) and bovine insulin (29%, IQR: 20.3-61.5), respectively. Here, we demonstrate different binding characteristics between IAA and IA, suggesting different epitope specificities. The observation of a significantly lower insulin binding to the ''natural insulin analogs'' (bovine and porcine insulin) compared to human insulin in the IA-group is in support of the concept that insulin analogs are eventually less immunogenic.

Biochimica et Biophysica Acta (BBA) - General Subjects, 1976

Smnmary The dissociation of insulin from human insulin antibodies has been investigated using a technique that is rapid and does not require addition of excess unlabelled insulin. A slow (k~ = 2. 10-3 min-~) and a fast (k2 = 4. 10-2 min-1) dissociating antibody component were identified in all studies. These have been shown to correspond, respectively, to the high and low affinity antibody components of equilibrium binding studies. The range of k~ and k2 values and their response to temperature change is small. Insulin resistance and stability of diabetes are not related to properties of antibody dissociation. Dissociation is faster in the presence of high (6-850 nM) insulin concentration due to increased binding to the fast dissociating component without change in the dissociation rate constants. When incubation time is increased beyond achievement of maximal binding there is a time-dependent rise in binding to the slow dissociating component, with a concomitant fall in k l. The traditional concept that equilbibrium is established at maximum binding requires further examination. * To whom reprint requests should be sent.

MethodsX, 2016

The prevalence and high levels of anti-insulin antibodies (IA) have frequently been associated with brittle diabetes, lipodystrophy in the areas where the insulin is injected and/or poor metabolic control. When this happens the usual criterion adopted is the empirical change of insulin type and/or formulation intending to diminish the IA level and then to decrease the undesirable side-effects. Here, we present a rational two step radiometric method consisting in: A) a first-line radioligand binding assay (RBA) to assess IA in sera of these patients and detecting those with high levels. B) applying a displacement assay (RIA) to determine the in vitro cross-reactivity parameters (affinity constants and selectivity ratios) that quantify the relative degree of interaction between antibodies and alternative insulin analogs. [ 6 2 _ T D $ D I F F ] From these results we conclude that conventional criteria for selection of insulin analogs, in terms of pharmacokinetic and pharmacodinamic parameters, should be complemented with an appropriate test to assess affinity parameters when high IA title is demonstrated. [ 6 3 _ T D $ D I F F ] This manuscript introduces a rational method to determine the appropriated insulin replacement when high insulin antibodies levels are present. This protocol provides instructions and details in mathematical tools and laboratory processes for the analysis of serum samples. This method proved to be successful in a single case and requires confirmation using a large group of patients.

Diabetic Medicine, 1995

The influence of insulin binding antibodies on the pharmacokinetics of NPH insulin was studied in Type 1 diabetic patients on human insulin. Insulin-antibody binding (B,) was measured during a screening procedure in 155 Type 1 diabetic patients. In 36 patients, B, was <1.5 %, and in 38 patients B, was >10.0 %. Of these, 6 patients, group 1 (B, < 1.5 YO) and 8 patients, group 2 (B,, > 10.0 YO), respectively, subsequently participated in a pharmacokinetic study. Free insulin and the glucose infusion rate were measured using a euglycaemic clamp after subcutaneous injection of NPH insulin (0.4 U kg-'). The areas under the curve (AUC) of free insulin concentration were smaller for group 2 (p = 0.01) than for group 1 (212.2 & 22.0 vs 316.8 f 25.3 mU I-' h). The AUCs of the glucose infusion rate were also smaller for group 2 (p < 0.05) than for group 1 (2.50 f 0.32 vs 3.58 f 0.36 g kg-'). A significant negative correlation exists between the AUCs for free insulin concentration and insulin-antibody binding B, (r = 0.76, p = 0.001). The daily insulin dosage was higher in group 2 (p = 0.02) than in group 1 (0.66 f 0.03 vs 0.53 f 0.03 U kg-I). We conclude that insulin antibodies influence the pharmacokinetics of NPH human insulin. The demonstrable influence on the kinetics of free insulin and glucose utilization leads to a slight increase in daily total insulin requirements. KEY WORDS Antibody-bound insulin Free insulin Human NPH insulin Euglycaemic clamp Screening of Diabetic Patients One hundred and fifty-five Type 1 diabetic patients on human insulin for at least 6 months were screened for the presence of insulin antibodies. Mean age (SEM)

Diabetologia, 1986

In the presence of anti-insulin antibody, 2-to 3-fold enhancement of 1251-insulin binding to liver membranes was observed when binding was estimated by the radioactivity of 125I-insulin bound to the membrane pellets. However, after 125I-insulin was covalently cross-linked to liver membranes using disuccinimidyl suberate in the presence of anti-insulin antibody, sodium dodecyl sulfate-polyac~iamide gel electrophoresis and autoradiography showed that 12sI-insulin bound to the a-subunit of the insulin receptor was inhibited by antiinsulin antibody in an dose-dependent manner. More importantly, at an anti-insulin antibody dilution range between 1 : 50 and 1 : 5,000, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two 125I-labelled bands of tool wt 62,000 and 27,000, while only one band of tool wt 130,000 was revealed in the absence of anti-insulin antibody. These Mr=62,000 and Mr=27,000 bands were found to be the heavy and the light chain of anti-insulin IgG molecules respectively. Pepsin digested anti-insulin serum had only an in-hibitory effect on 125I-insulin binding to liver membranes. Non-immunized guinea pig serum or IgG completely abolished the enhanced effect of anti-insulin antibody. Further, this enhanced effect was inhibited by Fc fragment-specific anti-IgG serum or H&L-chain-specific anti-IgG serum in a dosedependent manner. Protein A also inhibited the effect of antiinsulin antibody. In IM-9 Iymphocytes and human red blood cell ghosts, which have no Fc7 receptors, enhancement of insulin binding was not observed in the presence of anti-insulin antibody. These data suggest that anti-insulin antibody-induced enhancement of insulin binding to liver membranes is not due to the enhanced binding to the insulin receptor itself but probably due to the binding of insulin-anti-insulin antibody complex to the Fcy receptor.

Diabetes, 1987

The binding profiles of insulin autoantibodies (IAA) and insulin antibodies (IA) to highly purified species variants and fragments of insulin were studied in direct immunospecific enzyme-linked immunosorbent assay (ELISA) and indirect absorption experiments with insulins covalently coupled to Sepharose beads. Five of 10 lAA-containing sera from insulin-naive nondiabetics that bound whole human (H) insulin did not bind whole porcine (P) or whole bovine (B) insulins. These sera bound H insulin B-chain but not P B-chain or desalanated P insulin, suggesting they were dependent on the presence of threonine B30. The other 5 lAA-containing sera bound H, P, B, and desalanated porcine insulins, but only 1 bound isolated B-chains. All 10 lA-containing sera from insulin-treated diabetics bound H, P, B, and desalanated P insulins, but only 1 bound to human (and porcine) B-chain. Further binding studies with ovine, rabbit, rat, and guinea pig insulins confirmed the H (threonine B30) specificity of the 5 lAA-containing sera. B30 residues do not appear to be dominant, however, when insulin is administered exogenously. Instead, IA bind predominantly to Achain or conformational determinants involving both chains. Scatchard analysis of a representative H insulin-specific IAA serum suggested that it contained a single binding affinity, whereas analysis of a representative lA-diabetic serum suggested it contained several different affinities. Diabetes 36:66-72, 1987 I nsulin antibodies (IA) are commonly found in the sera of insulin-treated diabetics and occasionally cause the clinical symptoms of insulin resistance (1), allergy (2), or lipoatrophy (3). Such sera have been viewed as From the

Diabetes, 1985

The clearing of monoclonal and polyclonal and antiinsulin antibodies from homogenous solutions at 100,000 × g was used to estimate the size of soluble insulin-antibody complexes at physiologic concentrations. Monoclonal antibodies cleared as a uniform population of 6.6 S independent of the insulin concentration. Polyclonal antibodies cleared as 6.6 S monomers at saturation and as 10 S particles when the amount of insulin bound decreased, suggesting that a soluble complex with two antibodies was formed. An increase of the affinity and a decrease of antibody valency can be related to the complex formation. The binding affinity of polyclonal sera depends on the composition of the affinities of the IgG monomers and on their ability to form 10 S complexes. The formation of insulin-antibody dimers precludes cross-linking and precipitation. Both types of insulin-antibody complexes have been found in the sera from patients treated with bovine insulin.

Diabetes, 1986

To elucidate the immune aspects of insulin-dependent diabetes mellitus (IDDM), we attempted to generate human monoclonal anti-insulin antibodies by fusing peripheral blood lymphocytes obtained from 10 insulin-treated IDDM patients with cells from a human lymphoblastoid cell line. Hybridomas that secreted immunoglobulins appeared in 9 of 400 wells. One of these hybridomas secreted anti-insulin antibody of the IgM class. The lymphocytic partner of this hybridoma was obtained from an IDDM patient who had undetectable levels of antibodies to insulin in his serum. Thus, by employing the hybridoma technique, it was possible to reveal the presence of insulin-sensitized B-lymphocytes in a patient who was serologically negative for anti-insulin antibodies. The monoclonal antibody recognized intact human insulin and insulins of other species, but not isolated A-and B-chains. This indicates that the antibody was functionally an autoantibody directed to an epitope formed by the native conformation of a highly conserved portion of the insulin molecule. This is the first report of a human hybridoma antibody to insulin. DIABETES 1986; 35:68-73. T he formation of anti-insulin antibodies in diabetes patients treated with insulin is well documented. 1 Insulin resistance, insulin allergy, and lipoatrophy are among the clinical problems attributed to antiinsulin antibodies. 14 Therefore, much attention has been focused on anti-insulin antibodies in diabetes. As homogeneous subpopulations of antibodies can provide us with information not revealed by heterogeneous antibodies, we set out to produce human anti-insulin antibodies as monoclonal From the Departments of Internal Medicine (A.L.

Diabetes Care, 2005

OBJECTIVE—To assess the impact of the development of high- or low-affinity insulin antibodies (IABs) on postprandial glucose tolerance, duration of insulin action, and clinical safety in patients with type 1 diabetes receiving inhaled insulin (Exubera). RESEARCH DESIGN AND METHODS—This study consisted of a prospective, randomized, open-label, parallel-group trial in which 47 patients with type 1 diabetes received NPH insulin twice daily plus either premeal inhaled insulin (INH group; n = 24) or premeal subcutaneous regular insulin (SC group; n = 23) for 24 weeks. Meal challenge and euglycemic clamp studies were performed on consecutive days at baseline, week 12, and week 24. Adverse events were monitored. RESULTS—For the INH and SC groups, mean (±SD) IAB levels were 3.5 ± 3.9 and 2.6 ± 4.1 μU/ml at baseline, respectively, compared with 101.4 ± 140.4 and 4.3 ± 9.4 μU/ml at week 24. At week 24, the changes from baseline were similar for the INH and SC groups for maximal plasma glucose...