Paul Zev Zimmet

The ethnic distribution of antibodies to glutamic acid decarboxylase: Presence and levels in insulin-dependent diabetes mellitus in Europid and Asian subjects

Our objective was to ascertain the frequency of antibodies to glutamic acid decarboxylase (GAD) in Europids and four Asian ethnic groups with insulin-dependent diabetes mellitus (IDDM) to gain insight into why the prevalence and incidence of IDDM varies so widely among ethnic and/or geographically diverse population groups. The subjects in this study were Europid (n = 49), Japanese (n = 16), Thai (n = 7), Korean (n = 21), and Chinese (n = 13) persons with IDDM with a duration ranging from 5 to 14 years. There were similar numbers of healthy controls matched for each ethnic group. A validated radioimmunoprecipitation assay used GAD from pig brain radiolabeled with 125I using chloramine T. Islet cell cytoplasmic antibodies measured by indirect immunofluorescence were expressed as Juvenile Diabetes Foundation units. The prevalence of antibodies to GAD, compared with Europids (63%), was much lower in all Asian populations with IDDM: Japanese (31%), Thai (29%), Korean (5%), and Chinese (27%). The mean level of antibodies to GAD, however, among diabetics from each population who gave a positive reaction, was similar. For all groups, the prevalence of antibodies to GAD was much higher than that of islet cell cytoplasmic antibodies. Almost all IDDM subjects positive for islet cell antibodies had antibodies to GAD, but the converse did not hold. A radioimmunoprecipitation assay for antibodies to GAD applied to serum from subjects with IDDM in various ethnic groups showed that Europids with IDDM had a much higher prevalence of such antibodies than did Asians. This held for all ethnic groups, and particularly Koreans. Thus, among different populations, there may be etiologic heterogeneity of IDDM.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoantibodies to glutamic acid decarboxylase and phenotypic features associated with early insulin treatment in individuals with adult-onset diabetes mellitus

We investigated the association of serum antibodies to glutamic acid decarboxylase (GADab) with early start of insulin treatment (≤ 1 year from diagnosis, or ≤ 2 years from diagnosis) using data from a representative sample of 374 adult‐onset insulin‐treated individuals from the Tasmanian Diabetes Register. Furthermore, we examined whether this association was stronger than the phenotypic characteristics (age at diagnosis, sex, family history of diabetes, level of obesity, duration of diabetes) often used for diabetes classification. In this cohort, 35.9 % of males and 38.5 % of females were GADab positive. Within the first year from diagnosis, 78.4 % of GADab positive people compared to 44.0 % of GADab negative people (p < 0.001) had started insulin treatment. Univariate associations with insulin treatment ≤ 1 year from diagnosis included GADab positivity, no family history of diabetes, lower BMI for men, and GADab positivity and lower BMI for women. In multivariate models, significant associations with insulin treatment ≤ 1 year from diagnosis included a family history of diabetes (OR = 0.47, 95 % CI = 0.23–0.95) and GADab positivity (OR = 2.19, 95 % CI = 1.01–4.73) for men, but only GADab positivity (OR = 7.53, 95 % CI = 3.09–18.30) for women. Age at diagnosis was not associated with insulin treatment ≤ 1 year or ≤ 2 years from diagnosis for either sex. These findings indicate that a positive GADab test result is strongly associated with start of insulin treatment within 1 or 2 years from diagnosis, more so than characteristics such as level of obesity and age at diagnosis.

Diabetic sera react with the glutamic acid decarboxylase molecule in a dimeric-oligomeric form

Glutamic acid decarboxylase (GAD) is a major autoantigen in insulin‐dependent diabetes mellitus (IDDM). This was initially identified as a 64–65 kD molecule according to migration in gels after immunoprecipitation from pancreatic islets. We studied the antigenicity of two different radiolabelled preparations of GAD, derived either by affinity purification from porcine brain and known to contain GAD 65 and GAD 67, or by expression from a cDNA for human GAD 65 by rabbit reticulocyte lysate (RRL). Radiolabelled immunoprecipitated pellets from the reaction of potent antisera to GAD from patients with IDDM were examined by autoradiography after SDS–PAGE under reducing or non‐reducing conditions. Also, preparations of porcine brain GAD were ‘depleted’ of GAD by exposure to antisera, and then similarly re‐examined. Autoradiography of radiolabelled GAD either affinity purified from porcine brain, or expressed by RRL, showed that the immunoprecipitated protein migrated under non‐reducing conditions according to a Mr of ∼110–130 kD, corresponding to dimeric forms of monomeric GAD of ∼55–65 kD. Depletion by immunoprecipitation of this minor higher Mr component from preparations of GAD left, in the supernatant, an abundance of GAD of Mr 64–65u2003kD corresponding to monomer that was completely non‐reactive with potent IDDM sera. We conclude that IDDM sera react with the GAD molecule in a dimeric (or oligomeric) form. Our findings have general connotations for self‐tolerance and autoimmunity.

The plecomacrolide vacuolar-ATPase inhibitor bafilomycin, alters insulin signaling in MIN6 β-cells

Inhibition of endosomal acidification disturbs insulin signaling in both liver and adipose cells. In this study we used MIN6 β cells to determine whether bafilomycin, a potent inhibitor of the proton-translocating vacuolar ATPase, disrupts insulin signaling in islet β cells. Pretreatment of MIN6 cells with varying concentrations of bafilomycin according to a time course revealed concentration and time-dependent changes in phosphorylation of insulin receptor signaling components. Increased phosphorylation of insulin receptor (IR), IRS2 and Akt was prolonged at low bafilomycin concentrations (10 and 50 nmol/L), whereas at high concentrations (100 and 200 nmol/L) phosphorylation rapidly returned to basal levels or below. Akt activation was demonstrated by transient increases in phosphorylation of BAD, cytoplasmic retention of FoxO1 and increased preproinsulin mRNA. Bcl2 expression was also transiently increased but reduced after 30 min exposure to bafilomycin, and this coincided with reduced cell viability. Thus, in β cells inhibition of endosomal acidification by low concentrations of bafilomycin transiently increases insulin signaling, whereas high concentrations promote cell death. Bafilomycin and other agents that interfere with insulin signaling may contribute to diabetes development through disturbing homeostatic control of β cell growth.

Islet cell antibodies and other markers of autoimmunity and diabetes mellitus in Nauruans

SummaryAmong the population of Nauru there is a high prevalence of diabetes with onset in early adult life. To ascertain whether autoimmunity to islet cell antigens contributes to this diabetes, a population survey of serum autoantibodies was performed. Subjects were grouped into euglycaemic control subjects, those with impaired glucose tolerance, and those with diabetes subdivided according to duration of disease. No subject was positive by immunofluorescence for islet cell autoantibodies. Various other autoantibodies to nuclear, thyroid and gastric autoantigens were detectable, at comparable frequencies in the three groups. This population study on Nauruan subjects selected to include those in the early phases of disease negates a contribution from islet cell autoimmunity, and thus supports the concept that the disease is the Type 2 (non-insulin-dependent) type.

Transplacental exposure to the vacuolar-ATPase inhibitor bafilomycin disrupts survival signaling in β cells and delays neonatal remodeling of the endocrine pancreas

A wave of beta cell apoptosis occurs around 2 weeks of age in rats and mice. We have previously reported that exposure in utero to bafilomycin, a plecomacrolide antibiotic that inhibits the vacuolar (v)ATPase enzyme and contaminates the human diet, delays this neonatal wave and accelerates diabetes in non-obese diabetic (NOD) mice. Here we exposed C57BL/6J mice in utero to bafilomycin and assessed the effects on islet morphology, apoptosis and activation of cell survival signaling in beta cells. The neonatal wave of beta cell apoptosis was associated with high expression and low phosphorylation of the pro-apoptotic Bcl-2 family protein Bad, whereas after weaning (3 weeks of age) Bad was down-regulated and beta cell apoptosis was low. In contrast, in bafilomycin-exposed mice the frequency of apoptotic beta cells and the expression of Bad remained high after weaning. Bafilomycin exposure also inactivated the insulin/IGF signaling pathway intermediate, FoxO1, and increased the insulin content in neonatal islets. Thus, exposure in utero to bafilomycin disrupts the regulation of Bad in neonatal beta cells, increases cell survival signaling and delays the neonatal wave of apoptosis. Increased expression of Bad in adult beta cells provides an explanation for accelerated diabetes in bafilomycin-exposed NOD mice, whereby disruption of neonatal islet-cell turnover may render the adult beta cells more susceptible to induced cell death.

Ability of GHTD-amide and analogs to enhance insulin activity through zinc chelation and dispersal of insulin oligomers.

GHTD-amide is a tetrapeptide originally isolated from human urine that has hypoglycemic activity. Insulin occurs in secretory granules of beta cells as zinc-stabilized hexamers and must disperse to monomeric form in order to bind to its receptor. The aim of this study was to identify whether GHTD-amide and an analog called ISF402 (VHTD-amide) reduce blood glucose through enhancement of insulin activity by dispersing oligomers of insulin. Peptides containing the HTD-amide sequence and a free alpha-amino group were optimal at binding Zn(2+) and adopting secondary structure in the presence of Zn(2+). Binding was concentration dependent and resulted in a 1:1 Zn:peptide complex. In vitro the tetrapeptides dispersed hexameric insulin to dimers and monomers. GHTD-amide and ISF402 potentiated the activity of hexameric insulin when co-injected into insulin resistant Zucker rats. Injection of peptides with insulin caused reductions in blood glucose and C-peptide significantly larger than achieved with insulin alone, and serum insulin time profiles were also altered consistent with a reduced clearance or enhanced dispersal of the injected insulin. Insulin potentiation by ISF402 was reduced when lispro insulin, which does not form zinc-stabilized hexamers, was used in place of hexameric zinc insulin. In conclusion, GHTD-amide and ISF402 are zinc binding peptides that disperse hexameric insulin in vitro, and potentiate the activity of hexameric insulin more so than monomeric lispro insulin. These results suggest that dispersal of hexameric insulin through chelation of Zn(2+) contributes to the hypoglycemic activity of these tetrapeptides.

Geographic Differences in Antibodies to Glutamic Acid Decarboxylase in Insulin-Dependent Diabetes Mellitus

Major ethnic and geographic differences exist for insulin-dependent diabetes mellitus (IDDM) around the world.1 The disease is most common in people of European descent (Europids) with the highest reported incidence in Scandinavian countries,1,2 particularly in Finland where the incidence exceeds 30 cases/annum per 100,000 population.3 The lowest incidence rates are in Asia, notably Korea at 0.5 cases/annum1 with low rates in Japan3 and China as well.4 These geographic and ethnic differences are possibly due to variations in environmental risk determinants, although genetic differences may also be operative.5

Metodos para el diagnostico de diabetes y de estados prediabeticos

A method for detecting autoantibodies to glutamic acid decarboxylase (GAD) in the serum of a patient as diagnostic of a diabetic or prediabetic condition in the patient, comprises contacting a serum sample from the patient with a GAD antigen and detecting binding of autoantibodies to GAD in the sample by the GAD antigen, wherein the GAD antigen comprises a GAD preparation containing an enhanced amount of dimer(s) or oligomer(s) of the 65 kD or 67 kD isoforms, or both, of GAD. A diagnostic kit is also inclosed.