GAD65 antibody prevalence and association with thyroid antibodies, HLA-DR in Chinese children with type 1 diabetes mellitus

Persistent humoral autoimmunity to the enzyme glutamic acid decarboxylase (GAD) has been described in a substantial proportion of patients with type 1 diabetes mellitus. Higher prevalence of GAD antibody in diabetes patients using a new radioligand-binding assay with recombinant human GAD65 antibodies (GAD65Ab) has been seen in several studies. Using this method, we have reassessed the prevalence of GAD65Ab and investigated the association of GAD65Ab with HbA1C values, C-peptide values, HLA-DR typing and thyroid autoimmune antibody in 70 Chinese children with type 1 diabetes mellitus (mean age of onset 8.21+/-3.84 years, mean duration 3.39+/-2.54 years). Our result revealed that GAD65 antibodies were present in 54.3% (38/70) of diabetes children. There was no significant difference in gender, diabetes onset and duration, HbA1c, C-peptide concentration and frequencies of HLA DR3, DR4, DR9, DR3/DR4, DR3/DR9 and DR4/DR9 genotypes between GAD65Ab+ and GAD65Ab- groups. There was no negative correlation between GAD65Ab values and duration of diabetes in those with GAD65Ab positivity (r=-0.239, P>0.05). The frequencies of antimicrosomal and anti-thyroglobulin antibodies in GAD65Ab+ (13.5,8.1%, respectively) were not different from GAD65- patients (9.4,12.5%, respectively).     

Therapy with GAD in diabetes

The enzyme glutamic acid decarboxylase (GAD) is of great importance for the neurotransmission in the central nervous system, and therefore of interest for treatment of pain and neurological disease. However, it is also released in pancreas although its role is not quite clear. GAD is a major auto‐antigen in the process leading to type 1 diabetes with both a clear cell‐mediated immune response to GAD and auto‐antibodies to GAD (GADA), which can be used as a predictor of diabetes. Administration of the isoform GAD65 can prevent autoimmune destruction of pancreatic beta cells in non‐obese diabetic (NOD) mice and the subsequent need for exogenous insulin replacement. In Phase I and II studies an alum‐formulated vaccine (Diamyd) has shown to be safe, and in a dose‐finding study in Latent Autoimmune Diabetes in Adults (LADA) patients 20‐µg was given subcutaneously one month apart indicating preservation of residual insulin secretion. A double‐blind randomized Phase II trial in 70 patients (10–18 years old) with recent‐onset type 1 diabetes showed significant preservation of residual insulin secretion and a GAD‐specific immune response, both humoral and cell‐mediated, but no treatment‐related adverse events. With this promising background further studies are on their way, both intervention in newly diagnosed type 1 diabetic patients, and trials to prevent the disease. Copyright © 2009 John Wiley & Sons, Ltd.     

Murine monoclonal glutamic acid decarboxylase (GAD)65 antibodies recognize autoimmune-associated GAD epitope regions targeted in patients with type 1 diabetes mellitus and Stiff-man syndrome

To study the immune response to glutamic acid decarboxylase (GAD) in insulin-dependent diabetes mellitus, monoclonal GAD antibodies after fusion of splenocytes from a nondiabetes-susceptible BALB/c mouse immunized with human recombinant GAD65 were generated. Of the 44 monoclonals, 35 are specific for the GAD65 isoform, whereas 9 also react with GAD67. Some 37 monoclonals, including all GAD65/67 reactive antibodies, react with GAD by Western blot analysis. The remaining 7 GAD65 monoclonals bind GAD only in an immunoprecipitation assay, which implies that they target epitopes dependent on the conformation of the GAD molecule. The¹²⁵I-GAD binding of the GAD65 monoclonals reactive on Western blotting was significantly diminished by all 3 sera from Stiff-man syndrome patients but only by 3/30 (10%) sera from type 1 diabetic patients. In contrast, the 7 monoclonal antibodies reactive with a conformation-dependent GAD epitope were competitive with 83% of GAD-autoantibody-positive sera from these diabetic patients. Using chimeric GAD65/67 proteins, the epitope region targeted by these monoclonals was mapped to the middle of GAD65 (amino acids 221–442). This central conformation-dependent GAD region was also targeted by sera from patients with type 1 diabetes. In conclusion, our data show that evne after common immunization of a nondiabetes-susceptible mouse strain, monoclonals were obtained which preferentially react with the GAD65 linear amino-terminus (amino acids 4–17) and a conformation-dependent region located in the middle of GAD targeted by autoantibodies, indicating that this GAD region is not restricted to the autoimmune response associated with the Stiff-man syndrome and the bete-cell destruction in type 1 diabetes mellitus.     

Significance of IA-2 antibody in Japanese type 1 diabetes: its association with GAD antibody

To investigate the presence and level of serum antibodies to IA-2 (IA-2A) in Japanese patients with adult type 1 diabetes in order to clarify its association with glutamic acid decarboxylase (GAD) antibody. Serum samples were obtained from 101 Japanese patients with type 1 diabetes, including 37 patients with slowly progressive form of type 1 diabetes. Serum levels of IA-2A and GADA were determined by radioimmunoassay. The study had a cross-sectional design. IA-2A and GADA were detected in 37 and 59% of these patients, respectively. Of the 37 slowly progressive form of patients, IA-2A and GADA were present in 49 and 86%, respectively (NS). GADA levels were significantly higher (P<0.05) in IA-2A positive than in IA-2A negative patients in slowly progressive form, but IA-2A levels did not differ significantly between GADA positive and GADA negative patients. Measuring IA-2A in combination with GADA is useful for the diagnosis and prognosis of type 1 diabetes in Japanese.     

Predictors of response to liraglutide in Japanese type 2 diabetes

AimIn Japan, liraglutide is approved for use alone or in combination with sulfonylureas, and the approved maximum dosage is 0.9 mg/day. This restriction could limit the glucose-lowering effect of liraglutide in Japanese patients with type 2 diabetes mellitus (T2DM). This study was designed to identify predictors of response to liraglutide therapy at the approved dosage.MethodsThis observational retrospective study included 380 patients with T2DM who were treated with liraglutide alone or in combination with sulfonylureas at Diabetes Centers located in four geographically different areas of Japan. Binary logistic regression analysis was used to identify patient characteristics associated with discontinuation of liraglutide, while multiple regression and decision tree analyses were used to identify predictors of response to liraglutide therapy.ResultsFactors associated with discontinuation of liraglutide included high BMI, long duration of diabetes, and prior insulin therapy. Predictors of response to liraglutide therapy in patients who did not use insulin previously included previous use of few oral glucose-lowering agents and high baseline HbA1c level.ConclusionThe results suggest greater efficacy of liraglutide monotherapy or liraglutide-sulfonylurea combination therapy in patients with short duration of diabetes, non-insulin therapy, and low BMI and high HbA1c level at baseline.     

The response to short-term intensive insulin therapy in type 2 diabetes

Aim: Although a short course of intensive insulin therapy (IIT) can improve beta-cell function and glycaemic control in most patients with newly diagnosed type 2 diabetes (T2DM), the impact of this intervention in diabetes of longer duration has not been carefully studied. Thus, we sought to evaluate the effect of short-term IIT in patients with established T2DM.
Methods: Thirty-four patients, with diabetes of mean 5.9 ± 6.6 years duration, underwent 4–8 weeks of IIT, with 4-h meal test administered at baseline and at 1 day post-IIT. A positive clinical response was defined as fasting glucose < 7.0 mmol/l off any antidiabetic therapy at the latter test.
Results: A positive response was achieved in 68% (n = 23) of the subjects. At baseline meal test, the responders had lower glucose levels than the non-responders from 120 to 240 min (all timepoints p ≤ 0.0008) and higher late incremental area-under-the-C-peptide-curve (AUC_Cpep), particularly from 60 to 150 min (all p < 0.005). Beta-cell function (ratio of AUC_Cpep to AUC_gluc divided by HOMA-IR) was similar between the groups at baseline (median 54.1 vs. 51.3, p = 0.62) but after IIT was significantly higher in the responders (109.3 vs. 57.4, p = 0.009). At baseline, the strongest predictors of the change in beta-cell function were glucose levels between 180 and 240 min (all r = −0.5, p = 0.005) and incremental AUC_Cpep from 120 to 180 min (all r ≥ 0.66, p ≤ 0.0001), both reflecting late-phase insulin secretion.
Conclusions: The clinical response to short-term IIT is variable, consistent with the heterogeneity of T2DM. However, preserved late-phase insulin secretion may identify those patients who can benefit from this intervention with improved beta-cell function.     

Cell-mediated immune responses to GAD and beta-casein in type 1 diabetes mellitus in Thailand

We measured the cell-mediated immune response to GAD and bovine beta-casein in 38 type 1 and 37 type 2 diabetic patients who visited diabetic clinics or who were hospitalized in Bangkok, Thailand, and in 43 normal controls, by using a lymphoproliferation assay. Positive results against GAD were found in 29/38 (76.3%) type 1, 6/37 (16.2%) type 2 diabetic patients and 1/43 (2.3%) normal controls. Positive results against bovine beta-casein were found in 18/38 (47.4%), 5/37 (13.5%) and 1/43 (2.3%) of these subjects, respectively. The frequencies of the positive results and the magnitude of the responses to both antigens in type 1 diabetic patients were significantly higher than those in the other two groups (P<0.001). In addition, the prevalence of a positive lymphoproliferative response to these antigens in type 1 diabetic patients was higher than that of anti-GAD antibody positivity in the same group of subjects (26.3%). Thus, the prevalence of positive lymphoproliferative response to GAD in type 1 diabetic patients studied was higher than the prevalence of other autoimmune markers previously reported in type 1 diabetic patients in Thailand.     

Albuminuria response to very high-dose valsartan in type 2 diabetes mellitus

OBJECTIVE: Renin-angiotensin system blockade is now standard in the management of the patient with type 2 diabetes mellitus. We aimed to investigate whether high doses of valsartan, an angiotensin receptor blocker, are superior to conventional doses to reduce urinary albumin excretion rates (UAER) in such patients. PATIENTS AND METHODS: Three hundred and ninety-one hypertensive patients with type 2 diabetes mellitus and UAER 20-700 microg/min were randomized to 160, 320 or 640 mg valsartan. All received valsartan 160 mg for the first 4 weeks. Valsartan dose was then increased in two of three groups for 30 weeks. Overnight urine collections at baseline, 4, 16, and 30 weeks in triplicate were used to assess proteinuria. RESULTS: Comparable albuminuria reductions occurred in all groups at week 4 (P<0.001). Subsequently, a highly significant albuminuria fall occurred with valsartan 320 and 640 mg (P<0.001) versus a modest additional change with 160 mg (P=0.03). At week 30, twice as many patients returned to normal albuminuria with valsartan 640 mg versus 160 mg (24 versus 12%; P<0.01). High doses were well tolerated, with no dose-related increases in adverse events, including hypotension and hyperkalemia. CONCLUSION: High doses of valsartan reduced albuminuria more than the more commonly used 160 mg dose, apparently independent of blood pressure. Thus, at least in type 2 diabetes mellitus, higher doses of valsartan are required to optimize tissue protection than for blood pressure control.     

GAD antibodies in probands and their relatives in a cohort clinically selected for Type 2 diabetes.

AIMS: A subset of patients who present as if they have Type 2 diabetes have positive pancreatic autoantibodies, and have been referred to as having latent autoimmune diabetes in adults (LADA). We assessed the prevalence and clinical characteristics of patients with glutamic acid decarboxylase antibodies (GADA) in a cohort clinically selected for Type 2 diabetes and determined the presence of diabetes and GADA in their first-degree relatives. METHODS: GADA were measured in 2059 subjects, not known to be related, and clinically selected as having Type 2 diabetes for genetic studies. Clinical characteristics were compared in GADA positive and GADA negative subjects. Diabetes and GAD antibody status were compared in 208 first-degree relatives of GADA positive and GADA negative probands. RESULTS: Of the subjects, 136 (7%) were GADA positive. Compared with the GADA negative subjects, they were slimmer (P < 0.001), diagnosed at a younger age (P = 0.011) and progressed to insulin faster (P < 0.001). Thirty-three per cent of GADA positive subjects had a first-degree relative with diabetes compared with 42% of GADA negative subjects (P = 0.034). The overall prevalence of GADA was similar in the first-degree relatives of GADA positive and GADA negative probands (4 v 5%), and 19 of 22 (86%) diabetic relatives of GADA positive probands were GADA negative. CONCLUSION: Despite clinically selecting a Type 2 diabetes cohort, 7% were GADA positive with an altered phenotype. These GADA positive patients had a strong family history of non-autoimmune diabetes. This suggests that, in this subgroup of patients, autoimmune pancreatic beta-cell destruction occurs on a background of Type 2 diabetes genetic susceptibility.     

Higher prevalence of autoantibodies to insulin and GAD65 in Swedish compared to Lithuanian children with type 1 diabetes

We compared the prevalence of beta-cell autoantibodies and genetic risk factors in Sweden and Lithuania. Ninety-six patients from Sweden and 96 from Lithuania matched for age and gender (1-15 years old, median age 9.0 years) were included. We analyzed autoantibodies to insulin (IAA), glutamic acid decarboxylase (GADA) and the protein tyrosine phosphatase like IA-2 (IA-2A) as well as risk-associated polymorphisms of HLA, insulin and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) genes. The frequency of patients positive for IAA and GADA was higher in Sweden than in Lithuania (p = 0.043 and 0.032). The differences remained even when the patients were matched for HLA, insulin and CTLA-4 risk genotypes. Patients with low levels of IAA had higher levels of HbA1c and ketones at diagnosis. The frequency of the risk haplotype DR4-DQ8 was higher in Swedish than in Lithuanian patients (p = 0.004), as well as the high-risk combination of DR4-DQ8 and DR3-DQ2 haplotypes (p = 0.009). Our results suggest that autoimmune process against insulin and GAD(65) is more common at diagnosis in children in areas with high incidence of type 1 diabetes (T1D), independent of genetic risk markers. Furthermore, the disease in patients with insulin autoantibodies seems to be clinically milder.     

Association of sulfonylurea receptor 1 genotype with therapeutic response to gliclazide in type 2 diabetes

To investigate the effects of sulfonylurea receptor 1 (SUR1) exon 33 (TCC-->GCC, S1369A) polymorphism on responsiveness to gliclazide. About 115 patients with type 2 diabetes were treated with gliclazide for 8 weeks. SUR1 genotypes were tested by Taqman-PCR. After gliclazide treatment, there was association between T/G polymorphism and decrease of HbA1c. G carriers were more sensitive to gliclazide and the decrease of HbA1c was more significant than TT genotype (TT, 0.76%+/-1.70%; TG+GG, 1.60%+/-1.39%, P=0.044). The polymorphism of SUR1S1369A was associated with the therapeutic efficacy of gliclazide.     

Impaired chronotropic response to exercise in acute myocardial infarction patients with type 2 diabetes mellitus

This study was undertaken in acute myocardial infarction (AMI) patients with non-insulin-dependent diabetes mellitus (type 2 DM) to investigate their impaired chronotropic response to exercise. Seventy-one AMI subjects entered the study, 30 with type 2 DM and 41 age- and body mass index-matched non-DM (control) patients. One month after the onset of AMI, these patients underwent cardiopulmonary exercise testing on a treadmill under a ramp protocol. Anaerobic threshold (AT) and peak oxygen uptake (peak VO2) were determined as indicators of exercise capacity. Plasma norepinephrine (NE) concentration was measured in blood samples obtained at 2 time points: during pre-exercise rest and immediately after peak exercise. The change in NE concentration during exercise, as an index of sympathetic nervous activity, was calculated as a percentage: deltaNE = [(NE during exercise) - (resting value)]/(resting value) x 100. The change in heart rate (HR) during exercise was calculated as a simple difference: deltaHR = [(peak HR) - (rest HR)]. Index of chronotropic response to exercise was then quantified as the deltaHR/deltaNE during exercise. No significant intergroup differences in ejection fraction at rest or HR at peak exercise were observed. However, VO2 at AT, peak VO2, deltaHR, and deltaHR/deltaNE were significantly lower in the type 2 DM group than in the non-DM group. DeltaHR correlated with VO2 at AT (r = 0.49, P<0.001) and with peak VO2 (r = 0.53, P<0.001) in all subjects. Also, deltaHR/deltaNE correlated with VO2 at AT (r = 0.42, P<0.001) and with peak VO2 (r = 0.44, P<0.001) in all subjects. AMI patients with type 2 DM had impaired cardiopulmonary responses to maximal and submaximal exercise testing and impaired chronotropic response to exercise, even though their cardiac function at rest was similar to that of non-DM AMI patients. The data suggest that one mechanism of impaired cardiopulmonary response to exercise in AMI patients with type 2 DM groups is an impaired chronotropic response.     

Changes in adiponectin multimer distribution in response to atorvastatin treatment in patients with type 2 diabetes

Objective  Multimeric high molecular weight (HMW) forms of adiponectin were previously shown to be inversely associated with the extent of atherosclerosis in males and are down-regulated in patients with the metabolic syndrome and type 2 diabetes. In this study, potential influences of atorvastatin therapy on adiponectin multimer distribution were studied in patients with type 2 diabetes.
Design, patients and measurements  The effect of 40 mg atorvastatin on HMW, medium molecular weight (MMW), and low molecular weight (LMW) isoforms of adiponectin were investigated in 75 patients (23 females; 52 males) with type 2 diabetes in an 8-week-long, placebo-controlled and randomized study. Adiponectin multimeric isoforms were detected by Western blot analysis.
Results  After atorvastatin therapy the median serum concentration of HMW adiponectin increased significantly by 42·3% (1·68 vs. 2·39 µg/ml; P  < 0·001), while concentrations of MMW adiponectin and LMW adiponectin significantly decreased by 20·8% and 23·2%, respectively (MMW: 3·31 vs. 2·62 µg/ml, P  = 0·047; LMW: 0·56 vs. 0·43 µg/ml, P  = 0·033). Median total adiponectin levels were not significantly altered by atorvastatin treatment (6·0 vs. 6·2 µg/ml, P  = 0·898). Consequently, the HMW : total-adiponectin ratio significantly increased by 25·0% (0·40 vs. 0·50; P  = 0·013).
Conclusions  Atorvastatin therapy is associated with significant changes in adiponectin multimer distribution in patients with type 2 diabetes. Since total adiponectin levels were not affected by intervention, atorvastatin may shift adiponectin size towards the HMW form.     

Islet cell antibodies and glutamic acid decarboxylase antibodies, but not the clinical phenotype, help to identify type 1(1/2) diabetes in patients presenting with type 2 diabetes

This study was undertaken to determine which type 1 diabetes-associated autoantibodies and what clinical characteristics are most useful to identify patients with type 1(1/2) diabetes. We studied 125 patients, recently diagnosed clinically with type 2 diabetes for the presence of islet cell antibodies (ICA), insulin autoantibodies (IAA), antibodies to glutamic acid decarboxylase(GADAb), and IA-2a (IA-2Ab). Patients with a diagnosis of type 2 diabetes who met all of the following criteria at diagnosis were studied: age > or = 30 years, no history of ketonuria or ketoacidosis, and not requiring insulin treatment. Thirty-six patients (29%) were positive for at least 1 antibody. Thirty-two (26%) were ICA positive and 20 (16%) GADAb positive. Insulin autoantibodies and IA-2Ab occurred less frequently in 2 (1.6%) and 8 (6.4%) patients, respectively. There was no significant difference in the ages at diagnosis between the Ab(+) and Ab(-) patients, age in years (range) 47.2 (32 to 64) versus 51.2 (31 to 77), respectively, P =.06. Body mass index (BMI) was different in the 2 groups, with Ab(+) patients being less obese, BMI (range) 28.3 kg/m(2) (17.6 to 54.9) versus 32.0 kg/m(2) (19.2 to 68.8), respectively, P =.01. Clinical presentation of diabetes was more commonly symptomatic with polyuria and polydipsia in Ab(+) patients, while in Ab(-) patients, diagnosis was more often incidental, P =.002. However, more than 95% of patients overlapped in both age and BMI irrespective of antibody status. Similarly, 42% of Ab(+) patients had their diabetes diagnosed incidentally, while 29% of Ab(-) patients presented with polyuria and polydipsia. We therefore conclude that screening with antibodies, mainly ICA and GAD, but not age, BMI, or clinical presentation should be used to identify type 1(1/2) diabetes.     

Early epitope- and isotype-specific humoral immune responses to GAD65 in young children with genetic susceptibility to type 1 diabetes

OBJECTIVE: The pattern of the humoral immunity to disease-associated autoantigens may reflect the severity of the autoimmune disease process. The purpose of this study was to delineate the maturation of the humoral immunity to one of the main autoantigens in type 1 diabetes (T1D), glutamic acid decarboxylase (GAD65). DESIGN AND METHODS: Serum samples were obtained for the detection of epitope- and isotype-specific antibodies sequentially with short intervals from 36 young children with HLA-conferred genetic susceptibility to T1D starting from the first appearance of GAD65Ab. During prospective observation, ten children developed T1D. Antibodies were analyzed using biotinylated anti-human immunoglobulin (Ig) antibodies and chimeric GAD molecules in radio-binding assays. RESULTS: The immune response to GAD65 started as reactivity to the middle region and spread rapidly to the C-terminal region. IgG1 antibodies dominated among the isotypes from the first appearance of GAD65Ab, while other IgG subclasses were observed to a lesser extent. IgG4 antibodies emerged clearly as the last IgG subclass. A broad initial response comprising three to four IgG subclasses and the lack of an emerging IgG4 response during follow-up was associated with increased risk for progression to clinical diabetes (P<0.05). CONCLUSIONS: The humoral response to GAD65 epitope clusters is relatively uniform in young children, whereas there is conspicuous individual variation in IgG subclass responses except for IgG1. A narrow initial IgG subclass response to GAD65 and the emergence of IgG4 antibodies were characteristic of those who remained non-diabetic over the first few years of GAD65 autoimmunity.