A case of insulin resistant diabetes with possible antibodies to insulin receptors.

A case of a 19-year-old, non-obese female with insulin resistant diabetes mellitus and polycystic ovary syndrome was reported. The maximal insulin requirement attained 360 units per day, but a satisfactory control of diabetes did not follow. The patient's serum contained not only anti-insulin antibodies, but also possible anti-insulin receptor antibodies which were demonstrated by the 125I-insulin binding test using insulin receptors derived from human placental plasma membrane. The insulin resistance in this case was assumed to be caused primarily by possible blocking antibodies to insulin receptors and partly by anti-insulin antibodies because of the following observations. First, high serum free insulin (165 microunits/ml) without hypoglycemia indicates the presence of insulin resistance due to other factors than antiinsulin antibodies. Second, the titer of 125I-insulin binding capacity of serum was not unusually higher than those seen in chronically insulin-treated diabetics. Third, immunologically heterospecies insulin (fish insulin) was also ineffective. The clinical features such as absence of ketoacidosis and association with polycystic ovary syndrome resemble those of an unique diabetic syndrome reported previously though acanthosis nigricans and endogenous hyperinsulinemia were not found in this case. Her insulin resistance remitted spontaneously and over the next 18 months' observation, her diabetes remained regulated without insulin therapy.     

Heterogeneity and specificity of human anti-insulin antibodies determined by isoelectric focusing

Anti-insulin antibodies are present in the majority of insulin treated diabetics, and in some cases these antibodies have been found to be highly specific for limited epitopes on the molecule. To determine how the human response differs from that seen in inbred animals, we have examined the heterogeneity and specificity of human anti-insulin antibodies by isoelectric focusing (IEF). In addition, we have used human insulin to examine the extent of autoreactivity in the serum of subjects treated with animal insulins. The majority of diabetic sera exhibited complex IEF spectra that were composed of discrete bands and unresolved smears. Autoradiography using 125I-beef, pork, and human insulin revealed some affinity differences; however, the predominant antibodies were capable of binding all insulins, including human. These specificity studies were extended by comparing competitive inhibition with excess cold insulins, and sera with highly specific binding of the A chain loop of beef insulin were identified. The spectra by IEF of these highly specific sera were found to be variable. Our results indicate that the majority of insulin-treated diabetics develop a heterogeneous antibody response that is more complex than the response of inbred animals and includes reactivity with autologous insulin. Although infrequent, individuals having antibodies directed at limited regions of the molecule can be identified and will provide valuable tools for dissecting this complex response.     

Clinical examination of diabetic patients treated with monocomponent insulin manufactured by MP Polfa

An effect of replacing conventional forms of insulin by the monocomponent insulin manufactured by "Polfa" was studied in the group of 22 diabetics. The patients were followed up for 12 months. An effect of monocomponent insulin on daily requirement of insulin, levels of anti-insulin, monocomponent and pancreatic peptide antibodies, compensation of diabetes mellitus, and lipodystrophy were investigated. New insulin preparation decreased anti-insulin and pancreatic peptide antibodies level and markedly diminished lipodystrophy. However, daily insulin requirement, degree of diabetes mellitus compensation, and anti-proinsulin antibodies level remained unchanged.     

The metabolism of glucose in diaphragm muscle from normal rats, from streptozotocin-treated diabetic rats and from rats treated with anti-insulin serum

1. The metabolism of [U-¹⁴C]glucose by the isolated diaphragm muscle of normal rats, rats rendered diabetic with streptozotocin and rats with transitory insulin deficiency after an injection of anti-insulin serum was studied. 2. The incorporation of [¹⁴C]glucose into glycogen and oligosaccharides was significantly decreased in the diabetic diaphragm muscle and in the muscle from rats treated with anti-insulin serum. 3. Neither diabetes nor transitory insulin deficiency influenced the oxidation of glucose, or the formation of lactate and hexose phosphate esters from glucose. 4. Insulin fully restored the incorporation of glucose into glycogen and maltotetraose in the diabetic muscle, but the incorporation into oligosaccharides, although increased in the presence of insulin, was significantly lower than the values obtained with normal diaphragm in the presence of insulin.     

A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum

A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum is described. Guinea pig anti-insulin serum was diluted to various extents with nonspecific guinea pig serum and incubated with insulin. After incubation, free insulin was separated from insulin-anti-insulin antibody complex by treatment with dextran-charcoal. Anti-insulin antibodies in the complex were dissociated from insulin by incubation with 0.23 M HCl and inactivated. The amount of dissociated insulin was measured by sandwich enzyme immunoassay using anti-insulin IgG-coated polystyrene balls and affinity-purified anti-insulin Fab'-horseradish peroxidase conjugate. The detection limit of anti-insulin antibodies in guinea pig serum was 6.7 pg/assay or 150 ng/liter of serum. The present enzyme immunoassay was 10,000-fold more sensitive than the previously described enzyme immunoassay, in which insulin-coated polystyrene balls were incubated with diluted guinea pig anti-insulin serum and subsequently with rabbit (anti-guinea pig IgG) Fab'-horseradish peroxidase conjugate.     

Idiotypic determinants on human anti-insulin antibodies are cyclically expressed

Insulin-treated diabetics develop a heterogeneous antibody response to the protein hormone. To understand the repertoire of this diverse immune response, we developed monoclonal antibodies (Mab) that recognize idiotypic determinants on anti-insulin antibodies. Two Mab that recognize distinct idiotopes on anti-insulin antibodies were studied in detail. These idiotopes were present on IgG, but not IgM, anti-insulin antibodies from the proband and from four of 11 insulin-treated diabetics. The determinants recognized were distinct from conventional allotype (Gm) markers and were found in both Type I and Type II diabetics. The partial inhibition of anti-idiotope binding by beef insulin suggests that idiotopes related to the binding site for beef insulin may be recognized. When the presence of idiotope-positive molecules was determined sequentially during insulin therapy, in one patient, idiotope expression was found to be cyclical. In contrast, the total amount of anti-insulin antibody was constant, whereas idiotope expression varied. These data suggest that common V region determinants are employed in the anti-insulin response, and their variable expression may reflect dynamic regulation during chronic insulin therapy.     

Association of specific immune response to pork and beef insulin with certain HLA-DR antigens in type 1 diabetes.

To test the association of HLA-DR antigens with high-responder and low-responder status to either beef or pork insulin, insulin antibodies in diabetic sera were separated into those with average low and those with average high affinity and their insulin-binding capacities for each insulin determined. Significantly less binding of pork insulin by the high affinity antibodies occurred in the group of patients with DR3 antigens compared with those with DR4 antigens (p less than 0.01) and DR3/4 antigens (p less than 0.01). The difference in the binding capacity of beef insulin by the high affinity antibodies between the groups with DR3 and DR4 antigens was less pronounced but still significant. The high-responder status of DR3/4 antigens to pork insulin suggests that the gene or genes associated with HLA-DR4, and responsible for a high response to pork insulin, are dominant to genes associated with HLA-DR3 and a low response. If extended to human insulin and different HLA-DR and HLA-B antigen patterns, these finding should help in the therapeutic selection of the appropriate insulin and thus reduce the induction of an anti-insulin response in patients with diabetes.     

Anti-Insulin Immune Responses Are Detectable in Dogs with Spontaneous Diabetes

Diabetes mellitus occurs spontaneously in dogs. Although canine diabetes shares many features with human type-1 diabetes, there are differences that have cast doubt on the immunologic origin of the canine disease. In this study, we examined whether peripheral immune responses directed against islet antigens were present in dogs with diabetes. Routine diagnostics were used to confirm diabetic status, and serum samples from dogs with (N = 15) and without (N = 15) diabetes were analyzed for the presence of antibodies against islet antigens (insulin, glutamic acid decarboxylase, insulinoma-associated protein tyrosine phosphatase, and islet beta-cell zinc cation efflux transporter) using standard radioassays. Interferon-γ production from peripheral blood T cells stimulated by porcine insulin and by human insulin was tested using Elispot assays. Anti-insulin antibodies were detectable in a subset of diabetic dogs receiving insulin therapy. Pre-activated T cells and incipient insulin-reactive T cells in response to porcine or human insulin were identified in non-diabetic dogs and in dogs with diabetes. The data show that humoral and cellular anti-insulin immune responses are detectable in dogs with diabetes. This in turn provides support for the potential to ethically use dogs with diabetes to study the therapeutic potential of antigen-specific tolerance.     

Transient extreme insulin resistance in shock during diabetic ketoacidosis.

Transient extreme insulin resistance was encountered during an episode of diabetic ketoacidosis (DKA) in an insulin-treated diabetic patient. On admission, the plasma glucose level was 1241 mg dl-1 and arterial blood pH 6.895 with HCO3- 4.7 mEql-1. An intravenous bolus injection of 20 units, followed by continuous infusion of 20 units h-1 of short-acting regular human insulin, was instituted. Ischemic myocardial changes were noted on the initial electrocardiogram, therefore fluid replacement was limited to 1,000 ml of 0.9% saline solution in the first hour. As the plasma glucose level declined by only 203 mg dl-1 (41 mg dl-1 h-1) in the first 5 h, the insulin dose was doubled every 2 h. At hour 4, the patient developed circulatory shock which required vasopressor support and respiratory assistance. A plasma glucose level of 300 mg dl-1 was not achieved until the total dosage of insulin amounted to 91,580 units at hour 25. Insulin resistance was not observed from that point on. The patient had neither insulin antibodies nor anti-insulin receptor antibodies in serologic testing. The insulin binding characteristics of the patient's erythrocytes were similar to those from healthy controls both with and without experimental acidosis and with a high level of beta-hydroxybutyrate. Among multiple potential factors, the severe shock associated with DKA has been considered as a primary cause of the transient severe insulin resistance in this case.     

A correlation between microalbuminuria and anti-insulin antibodies in type I diabetics

A link between circulating anti-insulin antibodies and diabetic glomerulopathy has been suggested. This paper presents two different studies aiming to detect a relationship between incipient nephropathy (indicated by microalbuminuria) and anti-insulin antibodies. In 64 type I diabetics, overnight urinary albumin excretion during an exercise-test was found to be correlated with systolic blood pressure (r = 0.258 p less than 0.05), anti-insulin antibodies (r = 0.258 p less than 0.05), and glycosylated hemoglobin (r = 0.258 p less than 0.05) whereas no correlation was found among these three parameters. In another group of 80 type I diabetics, urinary albumin excretion during a standardized exercise-test was also correlated with anti-insulin antibodies (r = 0.360 p less than 0.001). In this latter group, diabetics with elevated (greater than 200 microU/ml) levels of anti-insulin antibodies had higher values of microalbuminuria after exercise (p less than 0.001) when compared to those with lower or undetectable levels, although they did not differ with respect to blood pressure and glycemic control. Therefore, we confirm preliminary reports indicating a statistical relationship between anti-insulin antibodies and microalbuminuria. We hypothesize that anti-insulin antibodies may be an additional factor of risk in the pathogenesis of early (reversible) stages of diabetic nephropathy.     

Very pure porcine insulin in clinical practice.

In a one-year follow-up study the insulin dose in diabetic patients using very pure porcine insulin was compared with that in patients using conventional preparations. The dose of insulin used to obtain diabetic control was reduced by 7% in 108 patients treated solely with very pure porcine insulin from the start of insulin treatment when compared with 108 matched patients who had received conventional insulins. In 117 patients whose treatment had been changed from conventional bovine or bovine-porcine insulin to very pure porcine insulin the dose was reduced by 9%. A further 511 patients receiving conventional insulins were examined for local cutaneous or subcutaneous abnormalities at insulin injection sites. Lipoatrophy was found in 49 of these patients (10%), but not in patients using very pure porcine insulin. The results confirm that very pure porcine insulin reduces the insulin dose needed to maintain diabetic control and may resolve or prevent local reactions such as lipoatrophy. Long-term advantages in reduced antigenicity to insulin and contaminating peptides remain to be established.     

Plant-based corosolic acid: Future anti-diabetic drug?

Diabetes is one of the nation's most prevalent, debilitating and costly diseases. For diabetes, frequent insulin treatment is very expensive and may increase anti-insulin antibody production, which may cause unwanted side effects. Corosolic acid may also have some efficacy in the treatment of diabetes, but without induction of anti-insulin antibodies. Recently, corosolic acid from Lagerstroemia speciosa L. leaf extracts has been reported to act via an indirect mechanism (unlike insulin) in animal experiments. The insulin-complementary anti-diabetic therapeutic value observed in these Japanese preliminary clinical trials has led to renewed interest in the biosynthesis of this compound. So far, there has been no clear evidence for a corosolic acid biosynthetic pathway in plants. This article provides possible roles of corosolic acid and hypothetical information on the biosynthetic pathway in plants.     

Comparison of insulin antibody levels during the first 3 years of treatment of adult diabetics with monocomponent porcine lente-insulin and single peak beef NPH insulin

Insulin antibody production was studied in two groups of 9 adult diabetics each, who were never treated before with insulin. One group received Monocomponent porcine lente insulin (MC) and the other group single peak beef NPH insulin, for 3 years. Insulin antibodies were evaluated by antibody-bound immunoreactive insulin (Abl) and by the labelled insulin binding capacity (IBC) which presented a significant correlation best fitted by a logarithmic curve. The patients treated with MC insulin developed significant levels of insulin antibodies, however, at lower levels and appearing later in comparison to those with beef NPH. Only 3 patients did not produce significant levels of insulin antibodies. The highest titers occurred after different lengths of treatment in the two groups of patients. Abl decreased after continuation of treatment particularly in the MC series. While in the MC-treated patients there was some positive correlation between the insulin dose and the level of Abl no significant correlation was found between the diabetes control and insulin antibody titers in both groups of patients.     

Secretion of triglyceride and ketogenesis by livers from spontaneous diabetic BB Wistar rats

Livers from male and female BB Wistar spontaneously diabetic rats were perfused in vitro to determine the effects of spontaneously occurring insulin-dependent diabetes on the metabolism of fatty acid. The secretion of triglyceride and the incorporation of [1-14C] oleic acid into perfusate and hepatic triglyceride was reduced by the diabetic state, whereas beta-hydroxybutyrate production and output of total ketone bodies were increased. The spontaneous diabetic Wistar rat clearly is a suitable model to study the derangements induced in lipid/plasma lipoprotein metabolism by the insulin-dependent diabetic state; the data obtained with this model confirm our earlier observations on experimental insulin deficiency induced with alloxan, streptozotocin, and anti-insulin serum.     

Alterations in lipid peroxidation,lipid profile,insulin sensitivity,and hepatic histopathological changes in diabetic rats following the treatment with Salvadora persica

We examined the effects of various partitions of Salvadora persica extract on lipid profile (LP), lipid peroxidation, and insulin sensitivity (IS) of diabetic rats. The rats were divided into normal control, diabetic control (DC), standard, sham, and test groups. The test groups were treated with an oral dose of 200, 400, and 600 mg/kg of crude, aqueous, and ethyl acetate partition of S. persica extract. After 21 days of experiment, the fasting blood glucose (FBS), LPs, lipid peroxidation, IS, liver enzymes levels, liver histopathology, and body weight alteration were evaluated. A significant decrease in FBS and lipid profile (except HDL) were observed in rats treated with various dose of extract compared with the DC rats ( P < 0.05). Treating diabetic rats with various extracts of S. persica meaningfully decreased the level of malondialdehyde ( P < 0.05). Animals treated with various dose of aqueous extract showed better results ( P < 0.01). On the basis of used indirect indexes to determine IS, all partitions of extracts showed anti-insulin resistance effects in diabetic rats. On the basis of our statistical analyzing, treating diabetic rats with all of the three extracts of S. persica decreased the elevated levels of alanine phosphatase, aspartate aminotransferase, and alanine transferase. Also, pathological changes in the liver tissue were reduced following treatment with the S. persica. In conclusion, our results give evidence that the S. persica extract, especially aqueous partition, has a healing effect on diabetes and can be considered as an alternative therapy for this disease.     

Cross-linking of insulin receptors to MHC antigens in human B lymphocytes: evidence for selective molecular interactions

Molecular interactions between insulin receptors and MHC antigens were investigated in human B cells. Two B lymphoblastoid cell lines, IM-9 and 526, chosen for their high insulin binding capacity, were found to express 15,000 and 25,000 insulin receptors per cell, respectively. Insulin receptors were labeled with a 125I-photoreactive insulin analogue, and all other surface proteins by lactoperoxidase-catalyzed radioiodination. Neighbor proteins were cross-linked with a cleavable homobifunctional reagent dithio-bis-(succinimidyl propionate) (DSP) and solubilized before immunoprecipitation by anti-HLA monoclonal antibodies. Gel analysis of the precipitated proteins showed that 90% of insulin receptors precipitable by anti-insulin receptor antibodies were precipitated by anti-class I antibodies (anti-heavy chain and anti-beta 2-microglobulin) after cross-linking with 2 mM DSP. In neither IM-9- nor 526 cells could HLA antigens be precipitated by anti-insulin receptor antibodies, suggesting that the concentration of class I antigens largely exceeds the concentration of insulin receptors at the cell surface. In 526 lymphocytes, class I MHC antigens were also found to adjoin class II antigens, since both molecules could be coprecipitated with anti-HLA A, B, C and with anti-HLA-DR antibodies after chemical cross-linking. Down-regulation of insulin receptors by chronic exposure of IM-9 cells to insulin did not affect the amount of MHC molecules present on the cell surface, and conversely, class I MHC molecules were internalized in 526 cells irrespective of the presence of insulin. These results thus show that insulin receptors and MHC antigens form multimolecular complexes in the plasma membrane of cultured human B cells. These interactions, which do not appear to influence the regulation of these proteins on the cell surface, may be involved in the mechanism of hormone signaling.     

Adenosine 3′,5′cyclic monophosphate in adipose tissue of diabetic rats

Normal male rats were made chronically diabetic by injection of alloxan or acutely diabetic by injection of anti-insulin serum. The concentration of cyclic AMP in epididymal adipose tissue was increased approximately 24 h after alloxan administration and up to 7-fold 72 h post-alloxan. Treatment of alloxan-diabetic rats with insulin for 4 h completely suppressed lipolysis but only partially suppressed cyclic AMP levels; 6 h following insulin treatment cyclic AMP levels were normal. When segments of the epididymal fat bodies were incubated in vitro the high cyclic AMP levels were not maintained but instead decreased spontaneously. Addition of insulin to the incubation media decreased lipolysis in tissues of diabetic rats to levels measured in tissues of normal rats and accelerated the decline in cyclic AMP levels but did not return cyclic AMP levels to normal. Rats rendered acutely insulin deficient by injection of anti-insulin serum showed increased plasma glucose and free fatty acid levels and increased adipose tissue free fatty acid, and cyclic AMP levels 30 min following injection of the antiserum. Plasma glucagon levels increased but not until 2 h following anti-insulin serum, thereby excluding the possibility that an increment in plasma glucagon is the primary stimulus for the acceleration of lipolysis in diabetes. These data are consistent with the view that control of adipose tissue cyclic AMP levels in situ is an important physiologic action of insulin.     

Autoimmune insulin syndrome in a patient with progressive systemic sclerosis receiving penicillamine.

The case of a patient with progressive systemic sclerosis, who developed hypoglycaemia and insulin autoantibodies, is described. Repeated blood glucose measurements showed levels less than 2.8 mmol/l. High immunoreactive insulin levels, with undetectable free insulin, led to the discovery of anti-insulin antibodies in the patient's serum. He had no history of exogenous insulin use and was receiving penicillamine treatment. A double mechanism for the autoimmune insulin syndrome in this case is proposed: acting in a patient with increased humoral immunoresponsiveness, penicillamine might have induced the development of insulin autoantibodies.     

A significant increase of lysophosphatidylinositol 4-phosphate with insulin in isolated rat fat cells

We studied the effects of insulin on the incorporation of 32Pi into phospholipids in rat fat cells. When the cells were treated with insulin, a new radioactive phospholipid was detected on thin layer chromatography. The substance migrated slower than phosphatidylinositol 4,5-bisphosphate and was hardly detectable in the absence of insulin. This effect of insulin was both time- and dose-dependent with half-maximal stimulation at 120 microU/ml. Pretreatment of insulin with anti-insulin antibody or the cells with anti-insulin receptor antibody inhibited the effect of insulin. The product of phosphatidylinositol 4-phosphate hydrolyzed by phospholipase A2 was coincided with the substance on thin layer chromatography. Quinacrine inhibited the formation of the substance in a dose-dependent manner. These results suggested that insulin stimulates the generation of lysophosphatidylinositol 4-phosphate through the insulin-receptor interaction.     

Insulin and rabbit anti-insulin receptor antibodies stimulate additively the intrinsic receptor kinase activity.

This paper describes the properties of rabbit polyclonal antibodies directed against purified human insulin receptor which strongly stimulate the intrinsic tyrosine kinase activity. The stimulatory effect of the antibodies on the kinase activity was obtained on the insulin receptor autophosphorylation as well as on the kinase activity towards a synthetic substrate. This stimulation is additive to that induced by insulin. Moreover, rabbit antibodies do not impair insulin binding. These data strongly suggest that antibodies and insulin act through separate pathways. This conclusion is reinforced by the differences observed on the phosphopeptide maps of the receptor's beta subunit whose phosphorylation was performed either in the presence of insulin or rabbit antibodies. Interestingly, these polyclonal antibodies can also induce an activation of the receptor autophosphorylation by interacting only with extracellular determinants. The anti-insulin receptor antibodies mimic insulin in their stimulatory effect on amino acid (AIB) uptake, but they have a different effect to that found on the kinase activity; the simultaneous addition of the antiserum and insulin failed to stimulate this amino acid transport over the level induced by a saturating concentration of hormone.