Presence Of Anti-insulin Antibodies Research Articles

Non-diabetic hypoglycemia

Glucose is the main substrate utilized by the brain, and therefore numerous counterregulatory mechanisms exist to maintain plasma glucose concentration. This makes it rare for hypoglycemia to develop in people who are not taking hypoglycemic drugs, such as insulin or sulfonylureas, for diabetes. The symptoms of hypoglycemia are nonspecific. The presence of Whipple’s triad is necessary for diagnosis. When symptoms occur spontaneously, the patient can be evaluated for hypoglycemia. If this is not possible, then a 72-hour fasting test or a mixed meal tolerance test can be performed to create conditions for symptoms to occur. Non-diabetic hypoglycemia is mainly divided into two main groups: insulin-mediated (hyperinsulinism) and insulin-independent. The main causes of hypoglycemia due to endogenous hyperinsulinism are insulinoma and islet cell hyperplasia (nesidioblastosis), post-bariatric surgery, and autoimmune hypoglycemia with the presence of anti-insulin antibodies. Other important causes of hypoglycemia include hypoglycemic drugs, non-islet cell tumors, hormonal deficiencies (primary adrenal insufficiency, anterior pituitary insufficiency), and critical illnesses (liver/kidney failure). In this article, we provide an overview of the pathogenesis and treatment of hypoglycemia.

1318-P: Characteristics of Autoimmune Thyroid Disease in African American and Caucasian Children with Type 1 Diabetes Mellitus

Background: Patients with type 1 diabetes mellitus (T1DM) are at increased risk of autoimmune thyroid disease (AIT). In the pediatric age group, the prevalence of AIT varies across a wide range (3-50%), depending on the population studied. There is very limited data describing the association of T1DM and AIT disease in African-American (AA) children. Objective: Characterize the association between T1DM and AIT in AA and Caucasian children presenting to the same medical center. Methods: After IRB approval, the medical records of 130 AA and 130 Caucasian children with T1DM presenting to the Children’s Hospital of Michigan were reviewed. Data collected included age, gender, ethnicity, presence of AIT evidenced by positive anti-TPO or anti-TG antibodies, TSH, free T4, anti-islet, anti-insulin and anti-GAD antibodies. Comparison was drawn between the two ethnicities in regards to the prevalence of AIT as well as predisposing factors for the onset of AIT. Results: 12.3% of AA with T1DM developed AIT versus 23.8% of Caucasian. This difference was statistically significant with a p-value of 0.023. In the Caucasian group, female was a risk factor for developing AIT (37.9% in females vs. 12.5% in males, p-value 0.001) while gender was not a significant risk factor for AA patients (p-value 0.790). The presence of anti-insulin antibodies was a risk factor for Caucasians (p-value 0.024) but not for AAs (p-value 0.185). The survival analysis showed mean time for onset of AIT in AA with T1DM is 15.1 years vs. 11.7 years in Caucasians (Log Rank p-value 0.025). Discussion: Our study shows a significantly lower prevalence AIT in AA compared to Caucasian children with T1DM. Female gender and anti-insulin antibody are risk factors for Caucasians but not in AAs. Differences could be secondary to different genes implication given the different genetic background. Further investigation can lead to a better understanding of T1DM and thyroid autoimmunity in the AA population. Disclosure M. El Bejjani: None. M. Zidan: None. H. Zidan: Speaker’s Bureau; Self; Novo Nordisk Inc.

Anti-insulin antibodies in dogs with naturally occurring diabetes mellitus

The presence of anti-insulin antibodies was determined by ELISA in serum samples from 30 diabetic dogs receiving bovine insulin therapy and 30 normoglycaemic dogs. Twenty of the diabetic dogs had significant reactivity to both bovine (heterologous) and porcine (homologous) insulin compared to control dogs. In contrast there was no significant difference between the two populations in reactivity to canine distemper virus (CDV) or canine thyroglobulin. The high degree of correlation between anti-bovine insulin and anti-porcine insulin antibodies suggested cross-reactivity which was confirmed by performing a competition ELISA, with antibody binding to bovine insulin inhibited by pre-incubating serum with porcine insulin. The insulin B-chain, rather than the A-chain was the most reactive component of the insulin molecule although in some cases, diabetics with antibody reactivity to whole insulin protein showed minimal reactivity to the individual subunits. The data suggest that treatment of diabetic dogs with bovine insulin can lead to anti-insulin antibody production. These antibodies cross-react with homologous insulin and recognise conformational as well as linear epitopes.

Autoimmune markers and neurological complications in non-insulin-dependent diabetes mellitus

To verify whether autoimmune markers related to nervous system structures and other autoimmunity indexes present in diabetes mellitus are associated with subclinical neuropathy, we examined 48 non-insulindependent diabetic patients with and without neuroelectrophysiological alterations. Nerve conduction velocity at the external sciatic-popliteal nerve, at the sural nerve, at the median and ulnar nerves level has been evaluated. Autoimmunity was investigated by evaluating glutamic acid decarboxylase (GAD-Ab), insulin (IAA), GM3, GD3 and GT1b gangliosides, pancreatic islet cell (ICA) and anti-nervous-tissue autoantibody presence. Nerve conduction velocities were decreased in 72.9% of diabetic patients. Anti-insulin antibodies were detected in seven non-insulin treated diabetic patients and in higher amount in subjects with (17.1%) than in those without (7.7%) asymptomatic neuropathy. Anti-GM3 antibodies were detected in four diabetic patients all of whom presented neurological complication. A significant correlation has been found between neurological damage and presence of anti-insulin antibodies ( p < 0.05). In the case of GM3 autoantibody, a similar result was obtained, but the data failed to reach statistical significance. Our data demonstrate that autoimmunity might play a role in the development of peripheral neuropathy.

Anti-insulin antibodies may cause hypoglycaemia following pancreas transplantation

Some pancreas transplantation (PTX) recipients experience hypoglycaemia after transplant. We studied the incidence and pattern of hypoglycaemic symptoms following PTX and later studied the glucose and pancreatic hormone response to Sustacal in patients with and without hypoglycaemia following PTX. In a survey of 54 PTX recipients who were 2 to 33 months post-transplant, 29 reported symptoms of hypoglycaemia at any time following their transplant. Of 12 patients who tried to document their blood glucose during any episode, 5 had a blood glucose less than 3.3 mmol/l, and another 5 documented a blood glucose from between 3.33 and 3.88 mmol/l. The patients reporting symptoms were less likely to be insulin resistant with longer time post-PTX, lower body mass index (BMI), and on lower doses of prednisone. Later, 12 patients with established, repeated episodes of hypoglycaemia following PTX were case-matched to PTX recipients without hypoglycaemic symptoms. Fasting glucose, free and total immunoreactive insulin (IRI), C-peptide, proinsulin and glucagon were measured following a 4 h Sustacal challenge. The hypoglycaemic group was further divided into those whose glucose rose after Sustacal (Hypo-High) and those whose blood glucose did not rise from baseline concentration (Hypo-Flat). The Hypo-High subgroup had a lower fasting free/total IRI ratio in addition to a greater increase in blood glucose after Sustacal compared with either Hypo-Flat or non-hypoglycaemic transplant recipients. The low free/total insulin ratio and hyperglycaemic response to Sustacal are consistent with the presence of anti-insulin antibodies, an established cause of hypoglycaemia. In summary, documented hypoglycaemia, while uncommon, may occur and be significant following PTX. Anti-insulin antibodies may be one of the many factors contributing to hypoglycaemia in this population.

Autoimmune insulin syndrome

Initially described in Japan, the autoimmune insulin syndrome is caused by the presence of anti-insulin antibodies in patients who have never received insulin. This syndrome accounts for spontaneous or reactive hypoglycaemia with very high levels of total immuno-reactive insulin. Discordance between the levels of immunoreactive insulin and C peptide indicate the possible presence of anti-insulin antibodies; this can avoid an incorrect diagnosis of insulinoma. These autoimmune hypoglycaemias often present a difficult diagnostic problem in distinguishing them from factitious hypoglycaemia. The course of the autoimmune insulin syndrome is usually favourable, with a spontaneous rapid diminution of the levels of anti-insulin antibodies. The reasons for the appearance of anti-insulin antibodies and the exact mechanisms of the hypoglycaemia remain uncertain. However, the frequent association of the autoimmune insulin syndrome with certain autoimmune diseases suggest a common immune dysfunction. Drugs containing a sulphydryl group have been implicated in the aetiology of this syndrome.

Development of insulin and epidermal growth factor receptors during the differentiation of rat preadipocytes in primary culture

An homogeneous cell population isolated from the inguinal tissue of 3-day-old rats is able to proliferate in primary culture. In the presence of a physiological concentration of insulin (1.5 nM) it converts into cells exhibiting the morphology and the biochemical characteristics of adipocytes. Insulin and epidermal growth factor (EGF) receptors were studied during both the exponential growth and the adipose conversion phases of these cells. Binding experiments with 125I-labelled peptides were performed directly in the culture dishes. The number of high affinity insulin binding sites increased, during the entire culture period studied, reaching 18 days after plating the value of 10 600 × 2360. Control cells (cultured in the presence of anti-insulin antibody) exhibited an increase of the concentration of insulin binding sites from no more than 500 sites/cell to 6880 ± 1710 sites/cell between dat 0 and 9 (corresponding to the exponential growth phase); this increase was followed by a rapid reduction in insulin receptors during the stationary phase. The density of EGF binding sites increased between day 0 and 4 (one cell cycle), whether the cells were maintained or not with insulin, and plateaued therafter. Mature adipocytes freshly isolated from the inguinal tissue of 3-day-old rats had no detectable EGF binding sites, but their content in high affinity binding sites for insulin was similar to that of cells complete adipocyte conversion in primary culture.

Insulin and insulin-like growth factor I both stimulate metabolism, growth, and differentiation in the postneurula chick embryo.

Chick embryos after 48 h of development (day 2) maintained in ovo provide an adequate model to study hormonal influences in early organogenesis in vertebrates. In previous studies at this (prepancreatic) stage of chick embryogenesis we demonstrated not only the presence of an insulin-related material but also insulin receptors and insulin-like growth factor I (IGF-I) receptors. Further, when embryos developed in the presence of antiinsulin antibodies, we showed retardation in both morphological and biochemical events which strongly suggested a physiological requirement for insulin in normal embryogenesis. In the present study we have evaluated the effects of insulin, proinsulin, desoctapeptide insulin, and IGF-I when applied to day 2 chick embryos. At day 4 of development biochemical indices were compared in treated vs. control groups. Insulin (10-100 ng/embryo) increased the content of protein, total creatine kinase, creatine kinase MB isozyme, triglycerides, cholesterol, phospholipids, DNA, and RNA, in a dose-dependent fashion. IGF-I had a lower potency than insulin in stimulating both metabolic and growth indices and was nearly equipotent in stimulating the creatine kinase MB content (marker of muscle differentiation). The high relative potency of insulin together with the effects of proinsulin (less than 15%) and desoctapeptide insulin (less than 10%) compared to insulin on the chick embryo, led us to infer that at low doses (nanograms per embryo) insulin stimulates developmental processes mainly through the insulin receptor, with the possible exception of muscle differentiation. The broad range of metabolic, growth, and differentiation indices stimulated by insulin and IGF-I in chick embryos, at a stage when specific receptors for both peptides are present, suggests that insulin and IGF-I may have a regulatory, complementary, or overlapping role in normal chick embryo early development.

Partial purification and characterization of an insulin-like material from spinach and Lemna gibba G3.

The existence in invertebrates, unicellular eukaryotes, and prokaryotes of materials that resemble several vertebrate peptide hormones led to the suggestion that these peptide messengers may have arisen earlier in evolution than had previously been thought. Consistent with this hypothesis, we describe here material in two plants, spinach and Lemma gibba G3, that is very similar to mammalian insulin, yet distinctive. In each of the early purification steps, which consisted of acidic methanol chloroform extraction and sequential chromatography on C-18 hydrophobic resin, Sephadex G-50, CM-Sepharose, and a short C-3 high performance liquid chromatography column, the immunoactive material from plants resembled the common vertebrate insulins. The protein nature of the material was suggested by its destruction by Pronase but not by the inactivated enzyme. In addition, on TSK chromatography it eluted in a position similar to that of insulin, i.e. equivalent to a protein of 6000 daltons. Using an isocratic high performance liquid chromatography system, the plant immunoactivity eluted earlier, and thus was more hydrophilic, than most of the common mammalian insulins, including pork insulin. The interaction of the plant material with anti-insulin antibodies in a radioimmunoassay was confirmed by using an affinity column of anti-insulin antibodies which adsorbed the plant immunoactivity at neutral pH, and released the material with acid elution. Using a quantitative double radioimmunoassay, the plant insulin-like material was distinguished immunologically from chicken insulin. Although the plant insulin-like material is clearly distinct from pork insulin chromatographically, and from chicken insulin immunologically, it resembles vertebrate insulins in its overall configuration. The plant insulin-like material bound to insulin receptors on IM-9 lymphocytes and stimulated glucose oxidation and lipogenesis in isolated adipocytes from young rats. The bioactivity was neutralized in the presence of anti-insulin antibodies, but not in the presence of normal guinea pig IgG. The role of this insulin-like material in plants is unknown but its existence is consistent with an early evolutionary origin of the insulin messenger peptide family. Alternatively we cannot exclude a later convergent development of this family or introduction of vertebrate DNA into plants.

Effects of anti-insulin antibody on insulin binding to liver membranes: evidence against antibody-induced enhancement of insulin binding to the insulin receptor.

In the presence of anti-insulin antibody, 2-to 3-fold enhancement of 125I-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-polyacrylamide gel electrophoresis and autoradiography showed that 125I-insulin bound to the alpha-subunit of the insulin receptor was inhibited by anti-insulin insulin 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 mol wt 62,000 and 27,000, while only one band of mol 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 inhibitory 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 dose-dependent manner. Protein A also inhibited the effect of anti-insulin antibody. In IM-9 lymphocytes and human red blood cell ghosts, which have no Fc gamma receptors, enhancement of insulin binding was not observed in the presence of anti-insulin antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

New powerful insulin-like protein from human promyelocytic leukemia cells.

Potent insulin-like activity was found in the conditioned medium of human promyelocytic leukemia (HL-60) cells. The conditioned medium of HL-60 cells at high density stimulated [3H]glucose incorporation into lipids in rat adipocytes in a time- and dose-dependent manner. The dose-response curve for this factor was not parallel to that for insulin, and the maximal effect achieved was much greater than reached by insulin or multiplication-stimulating activity. Moreover, the maximal effect reached by either insulin or the conditioned medium was additive. The insulin-like activity was not suppressed in the presence of antiinsulin antibody. Insulin-like activity was not detectable by radioreceptor assay for insulin, suggesting that the factor does not act through the insulin receptor. The factor in the conditioned medium of HL-60 cells was heat stable and sensitive to trypsin. When the conditioned medium was subjected to gel filtration on a Sephadex G-100 column, the major part of insulin-like activity eluted in the position corresponding to an apparent molecular weight between RNAase and insulin markers. The remaining activity, approximately 10% of the total, appeared with a larger molecular weight species. On isoelectric focusing of the smaller molecular species, insulin-like activity was largely focused in the position corresponding to pI 7.8-8.2.

Modulation of binding and bioactivity of insulin by anti-insulin antibody: relation to possible role of receptor self-aggregation in hormone action.

Incubation of physiological concentrations of 125I-labeled insulin with liver membranes in the presence of anti-insulin IgG results in a 7- to 15-fold increase in the specific binding of the hormone. The low-affinity/high-capacity binding sites are replaced by an apparently homogeneous class of high-affinity sites, and the nonlinear Scatchard plots are converted to linear plots without a change in the maximum number of binding sites. Similarly, the binding of insulin to receptors in 3T3 fibroblasts is increased substantially in the presence of anti-insulin antibody, and the biological activity of subactive concentrations of insulin is enhanced by antibody in these cells. However, the affinity of 125I-labeled epidermal growth factors (EGF) in fibroblasts is not affected by anti-EGF IgG. In adipocytes anti-insulin IgG in the same concentration range only inhibits the binding of insulin and suppresses insulin-mediated glucose oxidation. Monovalent Fab' fragments from anti-insulin IgG inhibit the binding of the hormone, indicating that the enhancement of binding in liver membranes and fibroblasts requires the bivalency of the antibody.

Placental barrier to human insulin-I125 in insulin-dependent diabetic mothers.

Although the normal human placenta is impermeable to insulin, the effect of anti-insulin antibodies on this placental barrier in insulin-dependent diabetic mothers is not known. The placental transfer of human insulin-I125 was examined in 4 normal and 4 insulin-dependent diabetic mothers. Human insulin-I125 was infused at a constant rate for 60–90 min prior to delivery and immuno-precipitable insulin-I125 was measured in the maternal and umbilical venous plasma at delivery. In diabetic patients, insulin-I125 was separated from circulating endogenous anti-insulin antibodiesby acid alcohol extraction prior to immunoprecipita-tion. In normal mothers insulin-I125 radioactivitywas 683 ±110 cpm/ml (mean ± sem) in the maternal vein and 6 ± 2 cpm/ml in the umbilical vein. In three diabetic mothers with high plasma insulin binding capacities between 2580–8400 μU/ml, the umbilical venous insulin-I125 was 10 ± 10 cpm/ml when the maternal plasma insulin-I125 was maintained at 1388 ± 356 cpm/ml even though the antibodies were present in the fetal plasma. In another diabetic subject with low anti-insulin anti-bodies, no placental transfer of insulin-I125 was demonstrated. Thus in both normal and diabetic subjects, placental transfer of insulin-I125 is negligible and the presence of anti-insulin antibodies does not induce insulin transfer.

The effect of endogenous antibody on insulin-assay in the newborn infants of diabetic mothers.

Extract: The immunoassay of insulin in blood of infants born to diabetic mothers is complicated by the presence of anti-insulin antibodies. Twelve infants born to diabetic mothers receiving insulin up to the time of delivery (Group 1) and two born to diabetic mothers maintained by dietary restrictions without insulin (Group 2) were studied. Four infants born to normal mothers served as controls (Group 3). The range of values in cord blood of infants of Group 1 was 80-1000 (median 550) (method of HALES and RANDLE) and 33 to 72.5 % (median 53.8) ofthe radioactivity was bound to plasma proteins. In one infant in Group 1, maximum amount of the radioactivity (42.3 %) from labeled insulin (43 %) was bound to a γ-globulin (0% in Groups 2 and 3). In one infant in Groups 2 and 3 maximum amount of radioactivity was in the α-globulin fraction (44.1 and 35.3 % respectively). At 120 minutes after birth, glucose, 0.5 gm/kg body weight, was infused quickly into the umbilical vein of 6 patients in Group 1 and the two in Group 2. In the former, insulin levels rose significantly, 1- to 4-fold, despite the elevated level at commencement of infusion; in the latter, the insulin levels rose 4-fold in one case and 18-fold in the second. The amount of anti-insulin antibody present in serum of infants in Group 1 did not change significantly during the first 240 minutes after birth; neither was it affected by the administration of glucose and the subsequent appearance of endogenous insulin. Speculation: Although the infant born to a diabetic mother has elevated levels of insulin and circulating anti-insulin antibodies, his ability to release insulin following a glucose challenge appears unimpaired. This combination of abnormalities suggests an alteration in glucose homeostasis, but fails to explain the hypoglycemia commonly seen in such infants, unless the anti-insulin antibodies do not interfere with the hypoglycemic effects of insulin.