Autoantibodies against IA-2, GAD, and topoisomerase II in type 1 diabetic patients

Prevalence of autoantibodies against IA-2 (IA-2A), glutamic acid decarboxylase (GADA), and type II DNA topoisomerase (TopIIA) of Taiwanese type 1 diabetes mellitus (T1DM) patients was investigated. Correlations of these autoantibodies with patients' clinical manifestations were also analyzed. Prevalence of IA-2A, GADA, and TopIIA in our patients was 23.6%, 47.1%, and 55.2%, respectively. Eighty percent of the IA-2A recognized the carboxyl terminus of the IA-2 protein tyrosine phosphatase-like domain. Average disease duration of IA-2A+ patients was significantly shorter than that of IA-2A- patients [3.76+/-0.42 vs. 4.98+/-0.34 years, p = 0.028]. Presence of GADA was correlated with the mean age of onset [10.82+/-0.76 vs. 8.38+/-0.77 years for GADA+ and GADA- patients, p = 0.026]. Patients with adolescent onset have higher GADA prevalence and better residual beta-cell functions. TopIIA and GADA are suggested to be better markers for Taiwanese T1DM patients because of their higher prevalence and persistence.     

Analysis of GAD65 autoantibodies in Stiff-Person syndrome patients

Autoantibodies to the 65-kDa isoform of glutamate decarboxylase GAD65 (GAD65Ab) are strong candidates for a pathological role in Stiff-Person syndrome (SPS). We have analyzed the binding specificity of the GAD65Ab in serum and cerebrospinal fluid (CSF) of 12 patients with SPS by competitive displacement studies with GAD65-specific rFab-derived from a number of human and mouse mAbs specific for different determinants on the Ag. We demonstrate considerable differences in the epitope specificity when comparing paired serum and CSF samples, suggesting local stimulation of B cells in the CSF compartment of these patients. Moreover, these autoantibodies strongly inhibit the enzymatic activity of GAD65, thus blocking the formation of the neurotransmitter gamma-aminobutyric acid. The capacity of the sera to inhibit the enzymatic activity of GAD65 correlated with their binding to a conformational C-terminal Ab epitope. Investigation of the inhibitory mechanism revealed that the inhibition could not be overcome by high concentrations of glutamate or the cofactor pyridoxal phosphate, suggesting a noncompetitive inhibitory mechanism. Finally, we identified a linear epitope on amino acids residues 4-22 of GAD65 that was recognized solely by autoantibodies from patients with SPS but not by serum from type 1 diabetes patients. A mAb (N-GAD65 mAb) recognizing this N-terminal epitope was successfully humanized to enhance its potential therapeutic value by reducing its overall immunogenicity.     

An analysis of the cross-reactivity of autoantibodies to GAD65 and GAD67 in diabetes

Background

Autoantibodies to GAD65 (anti-GAD65) are present in the sera of 70–80% of patients with type 1 diabetes (T1D), but antibodies to the structurally similar 67 kDa isoform GAD67 are rare. Antibodies to GAD67 may represent a cross-reactive population of anti-GAD65, but this has not been formally tested.

Methodology/Principal Findings

In this study we examined the frequency, levels and affinity of anti-GAD67 in diabetes sera that contained anti-GAD65, and compared the specificity of GAD65 and GAD67 reactivity. Anti-GAD65 and anti-GAD67 were measured by radioimmunoprecipitation (RIP) using ¹²⁵I labeled recombinant GAD65 and GAD67. For each antibody population, the specificity of the binding was measured by incubation with 100-fold excess of unlabeled GAD in homologous and heterologous inhibition assays, and the affinity of binding with GAD65 and GAD67 was measured in selected sera. Sera were also tested for reactivity to GAD65 and GAD67 by immunoblotting. Of the 85 sera that contained antibodies to GAD65, 28 contained anti–GAD67 measured by RIP. Inhibition with unlabeled GAD65 substantially or completely reduced antibody reactivity with both ¹²⁵I GAD65 and with ¹²⁵I GAD67. In contrast, unlabeled GAD67 reduced autoantibody reactivity with ¹²⁵I GAD67 but not with ¹²⁵I GAD65. Both populations of antibodies were of high affinity (>10¹⁰ l/mol).

Conclusions

Our findings show that autoantibodies to GAD67 represent a minor population of anti-GAD65 that are reactive with a cross-reactive epitope found also on GAD67. Experimental results confirm that GAD65 is the major autoantigen in T1D, and that GAD67 per se has very low immunogenicity. We discuss our findings in light of the known similarities between the structures of the GAD isoforms, in particular the location of a minor cross-reactive epitope that could be induced by epitope spreading.     

Familiality of metabolic abnormalities is dependent on age at onset and phenotype of the type 2 diabetic proband

To determine the impact of a family history of the common form of type 2 diabetes and the phenotype of the proband on anthropometric and metabolic variables in normoglycemic first-degree relatives, we studied 2,100 first-degree relatives of patients with the common form of type 2 diabetes (FH+) and 388 subjects without a family history of diabetes (FH-). All subjects participated in an oral glucose tolerance test to allow measurement of insulin secretion [30-min incremental insulin/glucose (I/G 30)] and insulin sensitivity [homeostasis model assessment (HOMA) of insulin resistance (IR)]. A subset participated in a euglycemic clamp (n = 75) and an intravenous glucose tolerance test (n = 300). To study the effect of a particular phenotype of the proband, insulin secretion and sensitivity were also compared between first-degree relatives of diabetic probands with high and low waist-to-hip ratio (WHR) and probands with early and late onset of diabetes. FH+ subjects were more insulin resistant, as seen from a higher HOMA-IR index (P = 0.006) and a lower rate of insulin-stimulated glucose uptake (P = 0.001) and had more features of the metabolic syndrome (P = 0.02, P = 0.0002) compared with FH- subjects. Insulin secretion adjusted for insulin resistance (disposition index, DI) was also lower in the FH+ vs. FH- subjects (P = 0.04). Relatives of diabetic probands with a high WHR had reduced insulin-mediated glucose uptake compared with relatives of probands with a low WHR (P = 0.04). Relatives of diabetic patients with age at onset <44 yr had higher HOMA IR (P < 0.005) and lower DI (P < 0.005) than relatives of patients with age at onset >65 yr (highest quartile). We conclude that early age at onset of type 2 diabetes and abdominal obesity have a significant influence on the metabolic phenotype in the nondiabetic first-degree relative.     

The effect of etomoxir on insulin sensitivity in type 2 diabetic patients.

Oral application of 50 mg Etomoxir caused a significant rise (33.1%) of insulin-mediated glucose uptake. This was shown in a placebo-controlled, double-blind randomized study in 8 type 2 diabetic patients by using the euglycemic clamp technique. The mean metabolic clearance rate of glucose (MCR) was raised from 4.1 +/- 0.9 mg/(kg.min) to 5.4 +/- 1.2 mg/(kg.min) (x +/- SEM, P = 0.039). Plasma levels of free fatty acids (FFA), glucose counterregulatory hormones, lipids and C-peptide values during the clamps were not different after verum and placebo. We conclude that Etomoxir improves insulin sensitivity in type 2 diabetic patients.     

Oral Administration of Silkworm-Produced GAD65 and Insulin Bi-Autoantigens against Type 1 Diabetes

Induction of mucosal tolerance by oral administration of protein antigens is a potential therapeutic strategy for preventing and treating type 1 diabetes (T1D); however, the requirement for a large dosage of protein limits clinical applications because of the low efficacy. In this study, we generated a fusion protein CTB-Ins-GAD composed of CTB (cholera toxin B subunit), insulin, and three copies of GAD65 peptide 531–545, which were efficiently produced in silkworm pupae, to evaluate its protective effect against T1D. We demonstrate that oral administration of CTB-Ins-GAD suppressed T1D by up to 78%, which is much more effective than GAD65 single-antigen treatment. Strikingly, CTB-Ins-GAD enhance insulin- and GAD65-specific Th2-like immune responses, which repairs the Th1/Th2 imbalance and increases the number of CD4⁺CD25⁺Foxp3⁺ T cell and suppresses insulin- and GAD65-reactive spleen T lymphocyte proliferation and migration. Our results strongly suggest that the combined dual antigens promote the induction of oral tolerance, thus providing an effective and economic immunotherapy against T1D in combination with a silkworm bioreactor.     

Effect of short-term low-intensity exercise on insulin sensitivity, insulin secretion, and glucose and lipid metabolism in non-obese Japanese type 2 diabetic patients.

The aim of the present study was to investigate the effects of short-term physical exercise that did not change body mass on insulin sensitivity, insulin secretion, and glucose and lipid metabolism in 39 non-obese Japanese type 2 diabetic patients. Insulin sensitivity and insulin secretion were estimated with homeostasis model assessment insulin resistance (HOMA-IR) and HOMA-B-cell function proposed by Matthews et al., respectively. All patients were hospitalized and were engaged in low-intensity exercise that consisted of walking and dumbbell exercise for successive 7 days. There were no changes in hospital diet and the dose of any medications used throughout the study. Fasting glucose, insulin, and lipids were measured before and after exercise.After exercise, serum triglyceride levels significantly decreased, but no significant changes were observed in total and HDL cholesterol concentrations. Fasting glucose, insulin, and HOMA-IR levels significantly decreased after exercise, but HOMA-B-cell function did not change during the study. There was no significant difference between BMI levels before and after exercise.From these results, it can be concluded that short-term (7 days) low-intensity physical exercise combined with hospital diet reduces serum triglycerides, insulin resistance, and fasting glucose levels without affecting BMI in non-obese Japanese type 2 diabetic patients.     

Decline in Titers of Anti-Idiotypic Antibodies Specific to Autoantibodies to GAD65 (GAD65Ab) Precedes Development of GAD65Ab and Type 1 Diabetes

The humoral Idiotypic Network consisting of antibodies and their anti-idiotypic antibodies (anti-Id) can be temporarily upset by antigen exposure. In the healthy immune response the original equilibrium is eventually restored through counter-regulatory mechanisms. In certain autoimmune diseases however, autoantibody levels exceed those of their respective anti-Id, indicating a permanent disturbance in the respective humoral Idiotypic Network. We investigated anti-Id directed to a major Type 1 diabetes (T1D)-associated autoantibody (GAD65Ab) in two independent cohorts during progression to disease. Samples taken from participants of the Natural History Study showed significantly lower anti-Id levels in individuals that later progressed to T1D compared to non-progressors (anti-Id antibody index of 0.06 vs. 0.08, respectively, p = 0.02). We also observed a significant inverse correlation between anti-Id levels and age at sampling, but only in progressors (p = 0.014). Finally, anti-Id levels in progressors showed a significant decline during progression as compared to longitudinal anti-Id levels in non-progressors (median rate of change: −0.0004 vs. +0.0004, respectively, p = 0.003), suggesting a loss of anti-Id during progression. Our analysis of the Diabetes Prediction in Skåne cohort showed that early in life (age 2) individuals at risk have anti-Id levels indistinguishable from those in healthy controls, indicating that low anti-Id levels are not an innate characteristic of the immune response in individuals at risk. Notably, anti-Id levels declined significantly in individuals that later developed GAD65Ab suggesting that the decline in anti-Id levels precedes the emergence of GAD65Ab (median rate of change: −0.005) compared to matched controls (median rate of change: +0.001) (p = 0.0016). We conclude that while anti-Id are present early in life, their levels decrease prior to the appearance of GAD65Ab and to the development of T1D.     

Epitope analysis of GAD65 binding in both cord blood and at the time of clinical diagnosis of childhood type 1 diabetes.

The GAD65 epitope immunoglobulin binding pattern in cord blood of children (n=37), who later developed type 1 diabetes at 3.2-14.9 years of age, was analyzed. First, the binding at diagnosis was compared with that in the cord blood serum. The next comparison was between the cord blood serum and the mothers' serum taken at delivery. Basal GAD65 binding levels were determined in Protein A Sepharose-based radiobinding assays with (35)S-labeled human and rat GAD65, rat GAD67 and GAD65/67 fusion proteins representing N-terminal (N), middle (M) and C-terminal (C) epitopes. In the first comparison, 28/37 children had GAD65 binding above 2.44 relative units (RU) (upper three quartiles), representing a marked increase from birth in the binding to human GAD65 (p<0.0001), rat GAD65 (p<0.0001), N- (p=0.04), M- (p<0.0001), C- (p=0.001), and M + C-epitopes (p<0.0001), but not to rat GAD67. At birth, 9/37 had GAD65 binding above 1.56 RU (upper quartile) demonstrating that their binding of human (35)S-GAD65 was higher in cord blood than in the mother (p=0.008). Higher cord blood binding was also observed for the N- (p=0.02) terminal epitope but not for rat GAD65, rat GAD67, and the remaining epitopes. These data suggest that differences in the epitope GAD65 binding between mother and child at birth are limited. In contrast, the epitope pattern at diagnosis differed from that at birth, supporting the view that disease-associated epitopes develop between birth and diagnosis.     

The heterogeneity of islet autoantibodies and the progression of islet failure in type 1 diabetic patients

Type 1 diabetes mellitus is heterogeneous in many facets. The patients suffered from type 1 diabetes present several levels of islet function as well as variable number and type of islet-specific autoantibodies. This study was to investigate prevalence and heterogeneity of the islet autoantibodies and clinical phenotypes of type 1 diabetes mellitus; and also discussed the process of islet failure and its risk factors in Chinese type 1 diabetic patients. A total of 1,291 type 1 diabetic patients were enrolled in this study. Demographic information was collected. Laboratory tests including mixed-meal tolerance test, human leukocyte antigen alleles, hemoglobinA1c, lipids, thyroid function and islet autoantibodies were conducted. The frequency of islet-specific autoantibody in newly diagnosed T1DM patients (duration shorter than half year) was 73% in East China. According to binary logistic regressions, autoantibody positivity, longer duration and lower Body Mass Index were the risk factors of islet failure. As the disease developed, autoantibodies against glutamic acid decarboxylase declined as well as the other two autoantibodies against zinc transporter 8 and islet antigen 2. The decrease of autoantibodies was positively correlated with aggressive beta cell destruction. Autoantibodies can facilitate the identification of classic T1DM from other subtypes and predict the progression of islet failure. As there were obvious heterogeneity in autoantibodies and clinical manifestation in different phenotypes of the disease, we should take more factors into consideration when identifying type 1 diabetes mellitus.     

Impact of smoking on the metabolic action of subcutaneous regular insulin in type 2 diabetic patients.

BACKGROUND AND AIMS: Smoking has repeatedly been associated with alterations in both insulin sensitivity and insulin absorption in type 2 diabetes, which should lead to differences in the pharmacokinetic (PK) and pharmacodynamic (PD) properties of regular insulin (RI). However, a direct comparison of the PK/PD-effects of RI has never been performed in these patients. Therefore, the aim of this exploratory study was to investigate the time-action profile of RI in a small group of smoking and matching non-smoking patients with type 2 diabetes using the euglycemic glucose clamp technique, which is seen as the gold standard for PD/PK investigations. MATERIAL AND METHODS: Nine smokers (more than 10 cigarettes per day) and nine non-smokers matched for gender, age, and BMI (without significant differences in HbA (1c), diabetes duration or blood pressure) were enrolled in the study. Patients' blood glucose was stabilized overnight at 7.2 mmol/l using a Biostator. Smokers were required to smoke one cigarette within ninety minutes prior to injection of 18 U RI s. c. in the morning. Glucose infusion rates (GIR) were registered for the subsequent 480 min. RESULTS: Injection of 18 U of RI resulted in significantly higher insulin concentrations in smokers compared to non-smokers, in particular in the later part of the experiment (Insulin-AUC (240-480) 10.5 +/- 2.3 (mean +/- SD) vs. 7.8 +/- 1.6 microU/ml/min, p < 0.05). This was also reflected in the PD results with a higher metabolic effect in smokers in the last four hours of the experiment (GIR-AUC (240-480) 0.9 +/- 0.4 vs. 0.6 +/- 0.3 g/kg, p < 0.05). Pharmacokinetic analyses revealed a trend towards a lower insulin clearance in smokers (1.1 +/- 0.2 vs. 1.4 +/- 0.4 l/min, p = 0.08). CONCLUSIONS: This pilot study conducted in a small group of patients with type 2 diabetes shows that regular insulin exhibits a longer-lasting rise in insulin concentrations and a higher metabolic effect four to eight hours after injection in smokers compared to non-smokers. This suggests that hyperinsulinemia in smoking type 2 diabetic patients is at least partly caused by a deterioration in insulin clearance.     

Association analysis of the AIRE and insulin genes in Finnish type 1 diabetic patients

Mutations in the autoimmune regulator (AIRE) gene cause a recessive Mendelian disorder autoimmune polyendocrinopathy syndrome type 1 (APS-1 or autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy). APS-1 patients develop multiorgan autoimmune diseases including type 1 diabetes (prevalence 12%). The AIRE protein controls the central tolerance induction in the thymus by regulating the expression levels of tissue-specific peripheral antigens, such as insulin. We hypothesized that the insulin gene (INS) polymorphisms together with the AIRE variations may predispose individuals to diabetes. The role of the AIRE gene was tested both independently and on the condition of the INS risk genotype in the Finnish type 1 diabetes sample. A total of 733 type 1 diabetic cases and 735 age- and sex-matched healthy controls were used in the analysis. Five common single nucleotide polymorphisms (SNPs) in the AIRE gene were selected from the public database (dbSNP). The −23HphI polymorphism was used as a surrogate marker for the INS gene promoter repeat. The five genotyped SNPs in the AIRE gene showed no evidence of association with type 1 diabetes. As expected, the INS gene polymorphism −23HphI was significantly associated with susceptibility to type 1 diabetes (P=6.8×10⁻¹², χ² test). When the subclass of patients carrying the homozygote genotype of the INS gene was used in the analysis, the AIRE polymorphisms showed no association with the disease. In conclusion, the AIRE gene does not seem to contribute to disease susceptibility in Finnish type 1 diabetic patients, whereas the insulin gene represents a notable risk factor for disease in this population.     

Reduced muscle carnosine content in type 2, but not in type 1 diabetic patients

Carnosine is present in high concentrations in skeletal muscle where it contributes to acid buffering and functions also as a natural protector against oxidative and carbonyl stress. Animal studies have shown an anti-diabetic effect of carnosine supplementation. High carnosinase activity, the carnosine degrading enzyme in serum, is a risk factor for diabetic complications in humans. The aim of the present study was to compare the muscle carnosine concentration in diabetic subjects to the level in non-diabetics. Type 1 and 2 diabetic patients and matched healthy controls (total n=58) were included in the study. Muscle carnosine content was evaluated by proton magnetic resonance spectroscopy (3 Tesla) in soleus and gastrocnemius. Significantly lower carnosine content (-45%) in gastrocnemius muscle, but not in soleus, was shown in type 2 diabetic patients compared with controls. No differences were observed in type 1 diabetic patients. Type II diabetic patients display a reduced muscular carnosine content. A reduction in muscle carnosine concentration may be partially associated with defective mechanisms against oxidative, glycative and carbonyl stress in muscle.     

甘精胰岛素在2型糖尿病围手术期对血糖控制的有效性、安全性和效益成本分析

目的:比较2型糖尿病围手术期患者使用甘精胰岛素和速效胰岛素控制血糖的有效性、安全性和效益成本分析.方法:对外科系统需要择期或限期手术的2型糖尿病患者随机给予甘精胰岛素组加速效胰岛素类似物或预混胰岛素类似物控制血糖.结果:两组患者达到了类似的空腹血糖控制,但甘精胰岛素组达标率更高、血糖达标时间短以及低血糖反应更少.尽管甘精胰岛素组患者胰岛素成本较高,但其糖尿病相关检查治疗成本、病房成本以及迭标总成本更低.结论:甘精胰岛素加速效胰岛素类似物可有效控制T2DM患者的围手术期血糖,缩短血糖达标时间,减少低血糖发生率低以及降低住院费用.     

Meal-dependent regulation of circulating glycated insulin in type 2 diabetic subjects.

There is mounting evidence that elevated circulating concentrations of glycated insulin play a role in insulin resistance in type 2 diabetes. This study evaluated the secretion of glycated insulin in response to enteral stimulation in type 2 diabetic subjects. Following a mixed meal (450 kcal; 44 % carbohydrate; 40 % fat; 16 % protein), glycated insulin rose 10-fold to peak (60 min) at 104.5 +/- 25.0 pmol/l (p < 0.001), representing 22 % total circulating insulin. The response paralleled early rises in insulin and C-peptide, which peaked at 90 min and were more protracted. Maximum glucose concentrations were observed at 50 min. These data indicate that type 2 diabetic subjects exhibit a rapid meal-induced release of glycated insulin from readily releasable pancreatic beta-cell stores, which might contribute to impaired glucose homeostasis following enteral nutrition.     

Estimations of muscle interstitial insulin, glucose, and lactate in type 2 diabetic subjects

Previous measurement of insulin in human muscle has shown that interstitial muscle insulin and glucose concentrations are approximately 30-50% lower than in plasma during hyperinsulinemia in normal subjects. The aims of this study were to measure interstitial muscle insulin and glucose in patients with type 2 diabetes to evaluate whether transcapillary transport is part of the peripheral insulin resistance. Ten patients with type 2 diabetes and ten healthy controls matched for sex, age, and body mass index were investigated. Plasma and interstitial insulin, glucose, and lactate (measured by intramuscular in situ-calibrated microdialysis) in the medial quadriceps femoris muscle were analyzed during a hyperinsulinemic euglycemic clamp. Blood flow in the contralateral calf was measured by vein plethysmography. At steady-state clamping, at 60-120 min, the interstitial insulin concentration was significantly lower than arterial insulin in both groups (409 +/- 86 vs. 1,071 +/- 99 pmol/l, P < 0.05, in controls and 584 +/- 165 vs. 1, 253 +/- 82 pmol/l, P < 0.05, in diabetic subjects, respectively). Interstitial insulin concentrations did not differ significantly between diabetic subjects and controls. Leg blood flow was significantly higher in controls (8.1 +/- 1.2 vs. 4.4 +/- 0.7 ml. 100 g(-1).min(-1) in diabetics, P < 0.05). Calculated glucose uptake was less in diabetic patients compared with controls (7.0 +/- 1.2 vs. 10.8 +/- 1.2 micromol. 100 g(-1).min(-1), P < 0.05, respectively). Arterial and interstitial lactate concentrations were both higher in the control group (1.7 +/- 0.1 vs. 1.2 +/- 0.1, P < 0. 01, and 1.8 +/- 0.1 vs. 1.2 +/- 0.2 mmol/l, P < 0.05, in controls and diabetics, respectively). We conclude that, during hyperinsulinemia, muscle interstitial insulin and glucose concentrations did not differ between patients with type 2 diabetes and healthy controls despite a significantly lower leg blood flow in diabetic subjects. It is suggested that decreased glucose uptake in type 2 diabetes is caused by insulin resistance at the cellular level rather than by a deficient access of insulin and glucose surrounding the muscle cell.     

Early and quantal (by litter) expression of insulin autoantibodies in the nonobese diabetic mice predict early diabetes onset

Aiming to study the early stages of type 1 diabetes phenotype, before insulitis appears, we measured insulin autoantibodies (IAA) between 3 and 5 wk of age in the NOD mouse (early-IAA (E-IAA)). We report that IAA are found as early as at 3 wk of age, at weaning, and their expression is a quantal phenotype. Maternal autoantibody status influences this early phenotype, because animals of litters issued from IAA-positive ante partum mothers develop E-IAA with a significantly higher incidence than animals issued from IAA-negative mothers. These E-IAA represent synthesized rather than transplacental autoantibodies, as evidenced by higher levels in many offspring compared with maternal IAA, and negative as well as positive offspring in the same litters and it correlates with early diabetes onset, defining the first autoimmune window in diabetes pathogenesis. Therefore, autoimmune processes leading to type 1 diabetes initiate early in life, are influenced by maternal autoantibody status, and can be revealed by the presence of IAA. Our data suggest that the mechanisms responsible for the breakdown of self-tolerance are subjected not only to genetic predisposition, but also to the physiological status of the mother. Pathological progression to autoimmunity is marked by the presence of immunological windows relating early steps with final disease onset.