Newly diagnosed latent autoimmune diabetes in adults (LADA) is associated with low level glutamate decarboxylase (GAD65) and IA-2 autoantibodies. Diabetes Incidence Study in Sweden (DISS).

A quantitative assay with microSepharose was used to determine GAD65Ab and IA-2Ab levels in 771 population-based patients diagnosed with diabetes mellitus at 15 to 34 years of age, and in 828 matched controls. Among the patients, 587 (76%) were classified with type I, 108 (14%) with type II, and 76 (10%) with unclassifiable diabetes. The levels above normal demonstrated a prevalence of GAD65Ab in 66% of type I diabetes, 50% of type II diabetes and 54% of unclassifiable patients and for IA-2Ab in 40%, 17% and 21%, respectively. Among the autoantibody-positive sera, the LADA patients had a lower GAD65Ab index (median 0.19, p < 0.0001) and IA-2Ab index (median 0.28, p < 0.0001) than the type I patients (median 0.37 and 0.66). Patients with unclassifiable diabetes had a GAD65Ab (median 0.43) or IA-2Ab (median 0.63) index which was not different from the type I diabetes patients. Our data demonstrate that young adult new-onset LADA patients have low level GAD65Ab and IA-2Ab. The low-level autoantibodies may signify a less aggressive beta-cell autoimmunity, which may explain why these patients are often classified with type II or non-insulin-dependent diabetes.     

High Titers of Autoantibodies to Glutamate Decarboxylase in Type 1 Diabetes Patients: Epitope Analysis and Inhibition of Enzyme Activity

ObjectiveAutoantibodies to glutamate decarboxylase (GAD65Ab) are found in patients with autoimmune neurological disorders or type 1 diabetes. The correct diagnosis of GAD65Ab-associated neurological disorders is often delayed by the variability of symptoms and a lack of diagnostic markers. We hypothesized that the frequency of neurological disorders with high GAD65Ab titers is significantly higher than currently recognized.MethodsWe analyzed GAD65Ab titer, GAD65 enzyme activity inhibition, and GAD65Ab epitope pattern in a cohort of type 1 diabetes patients (n = 100) and correlated our findings with neurological symptoms and diseases.ResultsOverall, 43% (43/100) of patients had detectable GAD65Ab titers (median = 400 U/mL, range: 142250,000 U/mL). The GAD65Ab titers in 10 type 1 diabetes patients exceeded the 90^th percentile of the cohort (2,000250,000 U/mL). Sera of these 10 patients were analyzed for their GAD65Ab epitope specificity and their ability to inhibit GAD65 enzyme activity in vitro. GAD65Ab of 5 patients inhibited the enzyme activity significantly (by 34-55%). Three patients complained of muscle stiffness and pain, which was documented in 2 of these patients.ConclusionsBased on our findings, we suggest that neurological disorders with high GAD65Ab titers are more frequent in type 1 diabetes patients than currently recognized. (Endocr Pract. 2013;19:663-668)     

Improved meal-related insulin processing contributes to the enhancement of B-cell function by the DPP-4 inhibitor vildagliptin in patients with type 2 diabetes.

The aim of this study was to evaluate the contribution of insulin processing to the improved meal-related B-cell function previously shown with the DPP-4 inhibitor vildagliptin. Fifty-five patients with type 2 diabetes (56.5+/-1.5 years; BMI=29.6+/-0.5 kg/m(2); FPG=9.9+/-0.2 mmol/l; HbA1c=7.7+/-0.1 %) were studied: 29 patients were treated with vildagliptin and 26 patients with placebo, both added to an ongoing metformin regimen (1.5-3.0 g/day). A standardized breakfast was given at baseline and after 52 weeks of treatment, and proinsulin related to insulin secretion was measured with C-peptide in the fasting and postprandial (over 4 h post-meal) states to evaluate B-cell function. The between-treatment difference (vildagliptin-placebo) in mean change from baseline in fasting proinsulin to C-peptide ratio (fastP/C) was -0.007+/-0.009 (p=0.052). Following the standard breakfast, 52 weeks of treatment with vildagliptin significantly decreased the dynamic proinsulin to C-peptide ratio (dynP/C) relative to placebo by 0.010+/-0.008 (p=0.037). Importantly, when the P/C was expressed in relation to the glucose stimulus (i.e., the fasting glucose and glucose AUC(0-240 min), respectively), the P/C relative to glucose was significantly reduced with vildagliptin vs. placebo, both in the fasting state (p=0.023) and postprandially (p=0.004). In conclusion, a more efficient B-cell insulin processing provides further evidence that vildagliptin treatment ameliorates abnormal B-cell function in patients with type 2 diabetes.     

Sustained normoglycemia and remission phase in newly diagnosed type I diabetic subjects. Comparison between continuous subcutaneous insulin infusion and conventional therapy during a one year follow-up

After onset of type I diabetes 7 diabetics were randomized to subcutaneous insulin pump treatment (CSII) (age 12 to 29 years, mean: 21 years) and 7 diabetics to conventional insulin treatment (CI) (age 14 to 28 years, mean: 21 years). HbA1, glycosylated serum proteins and mean blood glucose (MBG) as parameters of metabolic control were determined monthly. After 2 months both groups showed HbA1 values in the normal range. Mean MBG values were (mean +/- SD) 116 +/- 7 mg/dl for CSII and 118 +/- 14 mg/dl for CI. Residual insulin secretion was determined monthly by fasting C-peptide. After 14 days, 5, 7, 8 months fasting C-peptide values were significantly (P less than 0.05) higher in CI. After one year fasting C-peptide was comparable in both groups (CSII and CI mean: 0.06 nmol/l). The administered insulin dose was comparable in both groups with a 55% reduction of insulin dose after 5 months in CSII (0.35 +/- 0.15 U/kg/24 h) and in CI after 7 months (0.31 +/- 0.28 U/kg/24 h). After 12 months of insulin therapy about 60% of the initial insulin dose was injected in both groups. 1 patient on CSII (12 years) and 2 patients on CI (15, 28 years) showed a complete remission (for 3-9 months) with no exogenous insulin and normal HbA1 values. 50% of the patients had episodes where they did need less than 0.2 U/kg/24 h insulin to maintain optimal diabetic control (3 CSII, 4 CI). During the first year of insulin treatment in type I diabetes with CSII as well as with CI a comparable near normalisation of diabetic control could be achieved.     

Plasma C-peptide concentration in women with Type 1 diabetes during early and late pregnancy

Diabet. Med. 29, e361-e364 (2012) ABSTRACT: Aims There are previous suggestions of increased C-peptide concentration in women with Type?1 diabetes during pregnancy. Our aim was to re-evaluate the hypothesis of a pregnancy-induced increase by measuring plasma C-peptide concentration in women with stable blood glucose control under standardized fasting and meal-stimulated conditions. Methods Ten women with Type 1 diabetes; median age 31.1?years, median diabetes duration 19?years, median HbA(1c) 52?mmol/mol (6.9%) were admitted to a clinical research facility for two 24-h visits in early (12-16?weeks) and late (28-32?weeks) pregnancy. Women They ate standardized study meals - 80-g carbohydrate dinner, 60-g carbohydrate breakfast, and fasted between meals and overnight. Closed-loop insulin delivery maintained stable and comparable glycaemic conditions. Paired samples for plasma glucose and C-peptide were obtained. Results Plasma glucose levels were comparable in early (median 6.5?mmol/l; interquartile range 5.6-8.6) and late pregnancy (median 7.0?mmol/l; interquartile range 6.1-7.8; P?=?0.72). There was no change in fasting or meal-stimulated plasma C-peptide concentration from early to late pregnancy; mean difference 4.0?pmol/l (95%?CI -6.0 to 7.0; P?=?0.9). Four women had detectable C-peptide; peak (range) early vs. late pregnancy 48.5 (10-115) vs. 40.0?pmol/l (80-105); P?=?0.5, which was weakly associated with plasma glucose; R(2) =?0.15, P?相似文献   

The impact of insulin administration during the mixed meal tolerance test

Diabet. Med. 29, 1279-1284 (2012) ABSTRACT: Aims The mixed meal tolerance test is the gold standard measure of endogenous insulin secretion. Practical issues limit the routine clinical use of this test, including omitting insulin prior to the ingestion of a high-carbohydrate liquid mixed meal, which can result in marked hyperglycaemia. We aimed to assess whether insulin omission is necessary during the mixed meal tolerance test and whether fasting C-peptide was a practical alternative to the test. Methods Ninety-one adults with insulin-treated diabetes (Type?1 n?=?56, Type?2 n?=?35) underwent two mixed meal tolerance tests; one standard without insulin and one with the patient's usual morning insulin. Results The 90-min serum C-peptide was highly correlated in the standard mixed meal tolerance test and the test with insulin (r?=?0.98, P?相似文献   

Gliclazide mainly affects insulin secretion in second phase of type 2 diabetes mellitus.

We studied the effect of the acute administration of gliclazide at 160 mg on insulin release during hyperglycaemic clamps in 12 type 2 diabetes patients, age 50 +/- 9.0 years, diabetes duration 5.5 +/- 4.8 years, fasting blood glucose 9.6 +/- 2.1 mmol/L (means +/- SD). After a 210 min of hyperinsulinaemic euglycaemic clamp (blood glucose 4.6 +/- 0.14mmol/L), gliclazide or placebo (randomised, double-blind, cross-over) was administered; 60 minutes later, a hyperglycaemic clamp (4hr) at 8mmol/L was started. Plasma C-peptide levels increased significantly after the administration of gliclazide (increment 0.17 +/- 0.15 vs. 0.04 +/- 0.07 nmol/L, p = 0.024) before the clamp. After the start of the hyperglycaemic clamp, the areas under the curve (AUC) for insulin and C-peptide did not differ from 0-10 min (first phase) with gliclazide. However, second-phase insulin release (30-240 min) was markedly enhanced by gliclazide. AUC plasma insulin (30 to 240 min) was statistically significantly higher after gliclazide (12.3 +/- 13.9 vs. -0.56 +/- 9.4 nmol/L x 210 min, p = 0.022); similarly, AUC plasma C-peptide (30 to 240 min) was also higher: 128 +/- 62 vs. 63 +/- 50 nmol/L x 210 min, p = 0.002). In conclusion, in long-standing type 2 diabetes the acute administration of gliclazide significantly enhances second phase insulin release at a moderately elevated blood glucose level. In contrast to previous findings in mildly diabetic subjects, these 12 type 2 diabetes patients who had an inconsiderable first phase insulin release on the placebo day, only showed an insignificant increase in first phase with gliclazide.     

Insulin secretion and sensitivity in hyperthyroidism

To examine the effect of hyperthyroidism on carbohydrate metabolism, we studied glucose-stimulated insulin secretion and glucose utilization in 8 subjects with Graves' disease before and after treatment for hyperthyroidism and 8 age-, sex- and weight-matched normal subjects. Subjects with Graves' disease had significant elevated serum levels of thyroxine (24.81 +/- 2.44 micrograms/dl, mean +/- SEM) and triiodothyronine (459 +/- 5.5 ng/dl, mean +/- SEM). Simultaneous measurement of plasma glucose, serum insulin and C-peptide levels during fasting and every 30 minutes up to 180 minutes after 75 g oral glucose loading was determined. In addition, plasma glucose, serum insulin and serum C-peptide were measured during euglycemic glucose clamp with insulin infusion of 40 mU/m2 min-1. Mean fasting plasma glucose (P less than 0.05, serum insulin (P less than 0.005) and serum C-peptide (P less than 0.005) levels were significantly higher in the hyperthyroid patients. After glucose loading, the plasma glucose (P less than 0.05), serum insulin (P less than 0.05) and C-peptide (P less than 0.05) responses were significantly higher in hyperthyroid patients at all times up to 180 minutes. During euglycemic clamp studies, the steady-state serum insulin levels were identical in the two groups. The glucose disposal rate was lower in hyperthyroid patients before treatment (P less than 0.01) than in normal subjects. After thyroid function had been normalized for 2 to 4 weeks, the glucose disposal rate increased significantly (P less than 0.05), but was still significantly lower than those of normal subjects (P less than 0.05). Our data show that patients with Graves' hyperthyroidism manifest glucose intolerance, hyperinsulinemia and insulin resistance.     

Comparison of the subcutaneous absorption of insulin glargine (Lantus) and NPH insulin in patients with Type 2 diabetes.

The aim of this study was to compare the subcutaneous absorption characteristics of insulin glargine with NPH insulin in patients with Type 2 diabetes. In this single-dose, double-blind, randomized, two-way crossover study, 14 patients with Type 2 diabetes (aged 40-70 years) previously untreated with insulin were randomized to receive in a fasting state either a single subcutaneous injection of 0.3 U/kg 125I-insulin glargine or 0.3 U/kg 125I-NPH insulin. The disappearance of radioactivity was monitored for forty-eight hours. The median time for 25%, 50% and 75% of the radioactivity to disappear from the injection site was significantly longer for insulin glargine compared with NPH insulin (T75% 15.0 and 6.5 h, p=0.009; T50% 26.3 and 13.4 h, p=0.009; T25% 42.4 and 26.6 h, p=0.019, respectively). The mean residual radioactivity remaining at 24, 36 and 48 h after injection remained significantly higher than NPH insulin (54.4 and 27.9%, p=0.0001; 35.0 and 17.0%, p=0.003; 19.2 and 9.2%, p=0.01, respectively). Mean plasma glucose levels reached a minimum after 14.6 and 9 h in response to insulin glargine and NPH insulin, respectively. The subcutaneous absorption of insulin glargine in fasting Type 2 diabetes patients was significantly (2-3 times) slower compared with NPH insulin in patients with Type 2 diabetes. The slower absorption of insulin glargine correlated with the fall in plasma glucose levels over a 24 h period compared with the faster insulin absorption and more rapid decrease in plasma glucose levels observed in response to NPH insulin. Both insulin glargine and NPH insulin were well tolerated.     

Anti-idiotypic antibody specific to GAD65 autoantibody prevents type 1 diabetes in the NOD mouse

Overt autoantibodies to the smaller isoform of glutamate decarboxylase (GAD65Ab) are a characteristic in patients with Type 1 diabetes (T1D). Anti-idiotypic antibodies (anti-Id) directed to GAD65Ab effectively prevent the binding of GAD65 to GAD65Ab in healthy individuals. Levels of GAD65Ab-specific anti-Id are significantly lower in patients with T1D, leading to overt GAD65Ab in these patients. To determine the possible protective role of GAD65Ab-specific anti-Id in T1D pathogenesis, we developed the monoclonal anti-Id MAb 8E6G4 specifically targeting human monoclonal GAD65Ab b96.11. MAb 8E6G4 was demonstrated as a specific anti-Id directed to the antigen binding site of b96.11. MAb 8E6G4 recognized human antibodies in sera from healthy individuals, T2D patients, and T1D patients as established by ELISA. We confirmed these MAb 8E6G4-bound human antibodies to contain GAD65Ab by testing the eluted antibodies for binding to GAD65 in radioligand binding assays. These findings confirm that GAD65Ab are present in sera of individuals, who test GAD65Ab-negative in conventional detection assays. To test our hypothesis that GAD65Ab-specific anti-Id have an immune modulatory role in T1D, we injected young Non Obese Diabetic (NOD) mice with MAb 8E6G4. The animals were carefully monitored for development of T1D for 40 weeks. Infiltration of pancreatic islets by mononuclear cells (insulitis) was determined to establish the extent of an autoimmune attack on the pancreatic islets. Administration of MAb 8E6G4 significantly reduced the cumulative incidence rate of T1D and delayed the time of onset. Insulitis was significantly less severe in animals that received MAb 8E6G4 as compared to control animals. These results support our hypothesis that anti-Id specific to GAD65Ab have a protective role in T1D.     

High-dose oral vitamin C partially replenishes vitamin C levels in patients with Type 2 diabetes and low vitamin C levels but does not improve endothelial dysfunction or insulin resistance

Endothelial dysfunction is a hallmark of Type 2 diabetes related to hyperglycemia and oxidative stress. Nitric oxide-dependent vasodilator actions of insulin may augment glucose disposal. Thus endothelial dysfunction may worsen insulin resistance. Intra-arterial administration of vitamin C improves endothelial dysfunction in diabetes. In the present study, we investigated effects of high-dose oral vitamin C to alter endothelial dysfunction and insulin resistance in Type 2 diabetes. Plasma vitamin C levels in 109 diabetic subjects were lower than healthy (36 +/- 2 microM) levels. Thirty-two diabetic subjects with low plasma vitamin C (<40 microM) were subsequently enrolled in a randomized, double-blind, placebo-controlled study of vitamin C (800 mg/day for 4 wk). Insulin sensitivity (determined by glucose clamp) and forearm blood flow in response to ACh, sodium nitroprusside (SNP), or insulin (determined by plethysmography) were assessed before and after 4 wk of treatment. In the placebo group (n = 17 subjects), plasma vitamin C (22 +/- 3 microM), fasting glucose (159 +/- 12 mg/dl), insulin (19 +/- 7 microU/ml), and SI(Clamp) [2.06 +/- 0.29 x 10(-4) dl x kg(-1) x min(-1)/(microU/ml)] did not change significantly after placebo treatment. In the vitamin C group (n = 15 subjects), basal plasma vitamin C (23 +/- 2 microM) increased to 48 +/- 6 microM (P < 0.01) after treatment, but this was significantly less than that expected for healthy subjects (>80 microM). No significant changes in fasting glucose (156 +/- 11 mg/dl), insulin (14 +/- 2 microU/ml), SI(Clamp) [2.71 +/- 0.46 x 10(-4) dl x kg(-1) x min(-1)/(microU/ml)], or forearm blood flow in response to ACh, SNP, or insulin were observed after vitamin C treatment. We conclude that high-dose oral vitamin C therapy, resulting in incomplete replenishment of vitamin C levels, is ineffective at improving endothelial dysfunction and insulin resistance in Type 2 diabetes.     

Pancreatic B-cell function in non-insulin-dependent diabetes mellitus during successive periods of sulfonylurea and insulin treatment: serum C-peptide response to glucagon and urine C-peptide excretion

Serum C-peptide responses to glucagon and daily urine C-peptide excretion in successive periods of different treatment in two groups of patients with non-insulin-dependent diabetes mellitus (NIDDM) (mean interval between two tests less than 1 month) were compared. In group A patients (n = 8), the glycemic control was improved after transferring the treatment from sulfonylurea (SU) to insulin (fasting plasma glucose: SU: 192 +/- 47, insulin: 127 +/- 21 mg/dl, mean +/- S.D., p less than 0.01). Fasting serum C-peptide immunoreactivity (CPR) was significantly lower at the period of insulin treatment (SU: 1.93 +/- 1.01, insulin: 1.47 +/- 0.79 ng/ml, p less than 0.05), but there was no difference in the increase in serum CPR (maximal--fasting) (delta serum CPR) during glucagon stimulation in the two periods of treatment (SU: 1.70 +/- 0.72, insulin: 1.47 +/- 0.98 ng/ml). In group B patients (n = 7), there was no significant difference in glycemic control after transferring the treatment from insulin to SU (fasting plasma glucose: insulin: 127 +/- 24, SU: 103 +/- 13 mg/dl). Fasting serum CPR was significantly lower during the period of insulin treatment (insulin: 1.39 +/- 0.64, SU: 2.21 +/- 0.86 ng/ml, p less than 0.025), but delta serum CPR during glucagon stimulation still showed no significant difference between the two periods (insulin: 1.97 +/- 1.16, SU: 2.33 +/- 1.57 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Comparison of the prevalence of glutamic acid decarboxylase (GAD65) and gliadin antibodies (AGA) in a randomly selected adult estonian population.

We aimed to test the hypothesis that gluten might be associated with the development of islet cell autoimmunity. A random sample of 200 persons (87 males, mean age 42.4 years) from Estonia including one patient with type I diabetes mellitus was studied. IgG-type glutamic acid decarboxylase (GAD65) antibodies were determined using radioligand-binding assay and IgG/IgA-type gliadin antibodies (AGA) by enzyme-linked immunosorbent assay. Generic HLA-DRB1* alleles were analyzed using a polymerase chain reaction. Although our results revealed the highest GAD65Ab index and a high IgA-type AGA in a person with diabetes, no correlation between GAD65Ab and AGA values was revealed among the other 199 persons (p > 0.05). There were also no differences between test values among persons with and without different HLA-DRB1* alleles (p > 0.05). In the GAD65Ab assay, one person (0.5 %; 95 % CI: 0 - 1.5) out of 199 exceeded the 99(th) centile of the GAD65Ab index. In summary, the present study does not confirm the possibility that there is a relationship between the immune reactivity against GAD65 and gliadin, at least in persons without type I DM.     

Insulin sensitivity after maximal and endurance resistance training

The purpose of the study was to compare the effects of maximal resistance training (MRT) vs. endurance resistance training (ERT) on improvements in insulin levels and glucose tolerance in overweight individuals at risk of developing type 2 diabetes. Eighteen participants with baseline values suggesting impaired glucose tolerance were randomly assigned to 1 of 2 groups. Group 1 engaged in supervised MRT (Bernstein inverted pyramid system: 5 × 3-4, 60-85% 1 repetition maximum [1RM]), 3 d·wk(-1) over 4 months, whereas members of group 2 acted as controls. Later, group 2 engaged in supervised ERT (3 × 12-15, 45-65% 1RM), 3 d·wk(-1) over a 4 month period with the 2 prebaselines as controls. Both interventions consisted of 8 exercises that included the entire body. Glucose (fasting and 2-hour test), insulin and C-peptide measures were assessed from pre to post in both groups. The MRT led to reduced blood levels of 2-hour glucose (p = 0.044) and fasting C-peptide (p = 0.023) and decreased insulin resistance (p = 0.040). The ERT caused a significant reduction in the blood levels of insulin (p = 0.023) and concomitant positive effects on % insulin sensitivity (p = 0.054) and beta-cell function (p = 0.020). The findings indicate that both MRT and ERT lead to decreased insulin resistance in people with a risk of developing type 2 diabetes; MRT led to a greater increase in glucose uptake capacity (in muscles), whereas ERT led to greater insulin sensitivity, supporting the recommendation of both MRT and ERT as primary intervention approaches for individuals at a risk of developing type 2 diabetes.     

Biomarker potential of C-peptide for screening of insulin resistance in diabetic and non-diabetic individuals

Insulin resistance is a hallmark feature of type-2 diabetes mellitus (T2DM). We determined the homeostatic model assessment insulin resistance (HOMA-IR) and evaluated its association with C-peptide, insulin, fasting blood glucose (FBG) and glycated hemoglobin (HbA1c) in T2DM patients and non-diabetic subjects. This study comprised a total of 47 T2DM patients and 38 non-diabetic controls. Venous blood samples from all the subjects were collected and sera were analyzed for FBG, HbA1c, insulin and C-peptide using an autoanalyzer. HOMA-IR was calculated using the following equation: HOMA-IR?=?fasting insulin (µU/ml)?×?fasting glucose (mmol/L)/22.5. There was a significant increase in the levels of FBG and HbA1c in diabetic patients. Although the levels of C-peptide and insulin did not differ significantly between the two groups, a significant increase in HOMA-IR was observed in T2DM patients. Both insulin and C-peptide were significantly correlated with HOMA-IR. In conclusion, C-peptide may serve as a simple and convenient predictor of HOMA-IR.     

Activated lymphocytes in patients with newly diagnosed type 1 (insulin-dependent) diabetes mellitus

The expression of activation antigens (transferrin receptor, IL-2 receptor and Ia antigen) on circulating T lymphocytes from Japanese children with Type 1 diabetes was studied using five monoclonal antibodies (Ab), OKT9, anti-Tac Ab, OKIa1, anti-human HLA-DR Ab and OKT3. For detecting Ia positive T cells, the dual staining technique using OKT3 and anti-Ia antibody was employed. Four out of six patients (67%) with newly diagnosed Type 1 diabetes showed a raised level of either OKT9 or Tac positive cells when examined at diagnosis. These patients, however, rapidly lost these activation antigens after the insulin therapy was started. In contrast, in 32 long-standing patients, only 2 (6%) had a high percentage of OKT9 positive cells and none of them demonstrated Tac positive cells. One out of six newly diagnosed patients or three out of 21 long-standing patients had a significantly high percentage of Ia-positive T cells compared with normal subjects. In poorly controlled long-standing patients whose HbA1 value was higher than 14%, none of them had an increased number of activated lymphocytes. Therefore, it is unlikely that insulin deficiency and hyperglycemia were responsible for the changes observed in these studies. Activated lymphocytes might be related to activation of the immune system involved in pathogenesis of Type 1 diabetes.     

Development of glutamic acid decarboxylase 65 (GAD65) autoantibody assay using biotin-GAD65 fusion protein

We evaluated a biotin-glutamic acid decarboxylase 65 (GAD65)-based enzyme-linked immunosorbent assay (B-ELISA) to detect GAD65 autoantibodies (GAD65Ab) in 78 sera from individuals with newly diagnosed type 1 diabetes. The GAD65Ab index of patients with type 1 diabetes (mean value of GAD65Ab index of 1.891) was significantly higher than those in 50 sera from healthy control group (mean value of 0.068). The intra- and inter-assay coefficients of variation (CV) were calculated to be 1.042 and 10.703%, respectively. The specificity of the B-GAD65 ELISA was comparable to the standard radioimmunoassay (RIA) which is routinely used in the laboratory. We describe the optimal conditions of the binding kinetics from each assay-step for the detection of GAD65Ab using the WHO standard serum 97/550 as a model autoantibody serum. We concluded that incubation times of 15, 90, and 90 min for step 1 (pre-incubation of Biotin14-GAD65 with serum), step 2 (binding the Ab/Ag complex to NeutrAvidin plate), and step 3 (incubation with HRPO-anti-human IgG), respectively, along with human serum dilutions of 1:50, would provide an optimal assay signal within a relatively short timeframe.     

The insulinotropic effect of GIP is impaired in patients with chronic pancreatitis and secondary diabetes mellitus as compared to patients with chronic pancreatitis and normal glucose tolerance

BACKGROUND: The incretin effect is reduced and the insulinotropic effect of the incretin hormone glucose-dependent insulinotropic polypeptide (GIP) is abolished in patients with type 2 diabetes mellitus (T2DM). OBJECTIVE AND DESIGN: To evaluate the causality of this deficiency we investigated 8 patients with chronic pancreatitis (CP) and normal glucose tolerance (NGT) (fasting plasma glucose (FPG): 5.5 (4.5-6.0) mM (mean (range); HbA(1c): 5.8 (5.4-6.3) %) and 8 patients with CP and secondary diabetes not requiring insulin (FPG: 7.1 (6.0-8.8) mM; HbA(1c): 7.0 (5.8-10.0) %) during three 15-mM hyperglycaemic clamps with continuous iv infusion of saline, glucagon-like peptide-1 (GLP-1) or GIP. RESULTS: The initial (0-20 min) insulin and C-peptide responses were enhanced significantly in both groups by GLP-1 and GIP, respectively, compared to saline (P<0.05). In both groups GLP-1 infusion resulted in significantly greater insulin and C-peptide responses from 20-120 min compared with saline infusion. During GIP infusion the late-phase insulin response (20-120 min) was 3.1+/-1.0 fold greater than during saline infusion in the group of patients with CP and NGT (P<0.05), whereas there was no significant differences in patients with CP and DM. CONCLUSIONS: The lack of GIP amplification of the late insulin response to iv glucose develops alongside the deterioration of glucose tolerance in patients with CP, suggesting that the same may be true for the loss of the GIP effect in patients with T2DM.     

Glutamate decarboxylase and GABA in pancreatic islets: lessons from knock-out mice.

The GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) is expressed in pancreatic beta-cells and GABA has been suggested to play a role in islet cell development and function. Mouse beta-cells predominantly express the larger isoform of the enzyme, GAD67, and very low levels of the second isoform, GAD65. Yet GAD65 has been shown to be a target of very early autoimmune T-cell responses associated with beta-cell destruction in the non-obese diabetic (NOD) mouse model of Type 1 diabetes. Mice deficient in GAD67, GAD65 or both were used to assess whether GABA is important for islet cell development, and whether GAD65 is required for initiation of insulitis and progression to Type 1 diabetes in the mouse. Lack of either GAD65 or GAD67 did not effect the development of islet cells and the general morphology of islets. When GAD65-/-(129/Sv) mice were backcrossed into the NOD strain for four generations, GAD65-deficient mice developed insulitis similar to GAD65+/+ mice. Furthermore, at the low penetrance of diabetes in this backcross, GAD65-deficient mice developed disease at the same rate and incidence as wildtype mice. The results suggest that GABA generated by either GAD65 or GAD67 is not critically involved in islet formation and that GAD65 expression is not an absolute requirement for development of autoimmune diabetes in the NOD mouse.