In light of an occurring growth of elderly people a ffected by type 2 diabetes and recent observations indicating that type 2 diabetes may be a disease of the innate immune system, we evaluated whether signs of islet cell autoimmunity are associated with an abnormal glucose control, the presence of insulin requirement, or an activation of the acute-phase response in older individuals with type 2 diabetes. GAD65 and IA-2 autoantibodies along with the acute-phase response markers fibrinogen and C-reactive protein were tested in 196 serum samples from patients with type 2 diabetes and in 94 nondiabetic control subjects over the age of 6 5 years from the Pittsburgh cohort of the Cardiovascular Health Study. Of the diabetic patients, 12% (24 of 196) had autoantibodies against GAD65 and/or IA-2, a prevalence significantly higher than that found in nondiabetic individuals (1 of 94, 1.1%; P = 0.001). Type 2 diabetic patients who were positive for GAD65 and/or IA-2 autoantibodies (Ab + ), as compared with those negative for these autoantibodies (Ab -), had an abnormal oral glucose tolerance test (OGTT) (P = 0.03) before and a higher frequency of oral hypoglycemic treatment (P = 0.003) at the time of autoantibody testing. No differences were seen in the percentage of insulin requirement in the two groups. Moreover, a statistically significant increase in fibrinogen (P = 0.005) and C-reactive protein levels (P = 0.025) was found in t y p e 2 diabetic patients with high levels of GAD65 and/or IA-2 autoantibodies as compared with Ab -patients and control subjects. In conclusion, in type 2 diabetic subjects ≥65 years old, the presence of islet cell autoimmunity is associated with an impairment of the acute-phase insulin secretion, as revealed by an OGTT. A pronounced activation of the acute-phase response, found to be associated with islet cell autoimmunity, may in part explain this defect in insulin secretion. These findings not only have direct implications for adequate classification and treatment of diabetes in the elderly, but also for understanding the autoimmune/inflammatory mechanisms involved in the pathogenesis of hyperglycemia. D i a b e t e s 4 9 :3 2-38, 2000

The objective of this study was to determine whether antigenic determinants localized within the extracellular domain of the neuroendocrine autoantigen tyrosine phosphatase-like protein IA-2 are targets of humoral responses in type 1 diabetes (T1DM). Previous studies indicated that the immunodominant region of IA-2 is localized within its intracellular domain (IA-2ic; amino acids 601-979). We analyzed 333 subjects from the Children's Hospital of Pittsburgh study, 102 of whom progressed to insulin-requiring diabetes (prediabetics). Autoantibodies from these individuals were initially assayed for ICA512bdc (Barbara Davis Center amino acids 257-556; 630-979), IA-2ic (amino acids 601-979), and IA-2 full-length (amino acids 1-979) in addition to islet cell antibody (ICA), glutamic acid decarboxylase, 65-kDa isoform, and insulin autoantibodies. We identified an autoantibody response reactive with the extracellular domain of IA-2 that is associated with very high risk of T1DM progression. Relatives with no detectable autoantibodies against ICA512bdc (or IA-2ic) exhibited antibody responses against the IA-2 full-length peptide (log rank, P = 0.008). This effect was also observed in first-degree relatives who were positive for glutamic acid decarboxylase, 65-kDa isoform (log rank, P = 0.026) or at least two islet autoantibodies but were negative for ICA512bdc (log rank, P = 0.022). Competitive binding experiments and immunoprecipitation of the IA-2 extracellular domain (amino acid residues 26-577) further lend support for the presence of autoantibodies reactive with new antigenic determinants within the extracellular domain of IA-2. In summary, the addition of measurements of autoantibodies reactive with the IA-2 extracellular domain to assays geared to assess the progression of autoimmunity to clinical T1DM may more accurately characterize this risk. This has considerable implications not only for stratifying high diabetes risk but also facilitating the search for pathogenic epitopes to enable the design of peptide-based immunotherapies that may prevent the progression to overt T1DM at its preclinical stages.

No abstract

Markers of humoral islet cell autoimmunity, such as autoantibodies (AAs) against the 65-kDa isoform of GAD (GAD65), serve as determinants of risk for autoimmune diabetes. Despite the high prevalence of diabetes in U.S. racial and ethnic minority adult populations, little is known concerning the prevalence of GAD65 AA in these groups. We estimated the prevalence of GAD65 AA in 1,064 diabetic and 1,036 nondiabetic participants who were 40 -90 years of age from the Third National Health and Nutrition Examination Survey (NHANES III), which provides a representative ethnic sample of the U.S. diabetic population. The prevalence of GAD65 AA was higher in diabetic participants compared with nondiabetic participants in non-Hispanic whites (n ‫؍‬ 920; 6.3% vs. 2.0%; P ‫؍‬ 0.001) and non-Hispanic blacks (n ‫؍‬ 534; 3.7% vs. 1.3%; P ‫؍‬ 0.08) but not in Mexican Americans (n ‫؍‬ 646; 1.2% vs. 2.6%; P ‫؍‬ 0.18). Among diabetic non-Hispanic whites and non-Hispanic blacks, being GAD65 AA positive was associated with lower BMI and C-peptide (P < 0.05). These results may reflect the outcome of an autoimmune process leading to ␤-cell destruction/dysfunction in non-Hispanic white and nonHispanic black adult diabetic patients as it occurs in a similar manner in type 1 diabetes. Among diabetic Mexican Americans, the lower prevalence of GAD65 AA suggests a lower frequency of autoimmune-related diabetes.

The discovery of islet cell antibodies (ICAs) was the prelude to the understanding that type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease. The issue regarding whether or not the measurement of ICAs should be completely replaced by biochemical markers detecting islet autoantibodies (AAs) for the prediction of T1DM has been the subject of endless international debates. In light of this controversy, we assessed the current role of ICAs as a predictive marker for T1DM progression. We examined a cohort of 1484 first-degree relatives (FDRs) of T1DM probands from the Children's Hospital of Pittsburgh Registry. These relatives were consecutively enrolled between 1979 through 1984 and followed up to 22 yr. Serum obtained at the time of enrollment was assayed for ICAs, glutamic acid decarboxylase (GAD)65, insulin A (IA)-2 AA, and insulin AAs (IAAs). In FDRs who had ICAs in addition to GAD65 and IA-2 AAs, the cumulative risk of developing insulin-requiring diabetes was 80% at 6.7 yr of follow-up, whereas this risk in those with GAD65 and IA-2 AAs without ICAs was only 14% at 10 yr of follow-up (log rank: P < 0.00001). Cox regression analysis showed that diabetes risk was significantly associated with the presence of ICAs in both subjects with low titer and high titer GAD65 and IA-2 AAs. The addition of IAAs in GAD65 and IA-2 AA-positive relatives did not increase the cumulative risk for conversion to insulin-treated diabetes. We provide evidence that a subgroup of ICAs predicts a more rapid progression to insulin-requiring diabetes in GAD65 and IA-2 AA-positive relatives and should remain part of the assessment of T1DM risk for intervention trials. In addition, these findings provide impetus for efforts to identify a novel islet autoantigen(s) reactive with this ICA subset.

OBJECTIVEThe characterization of diverse subtypes of diabetes is a dynamic field of clinical research and an active area of discussion. The objective of this study was to identify new antigenic determinants in the neuroendocrine autoantigen IA-2 (ICA512) and assess whether circulating autoantibodies directed to new IA-2 epitopes identify autoimmune diabetes in young and adult populations with diabetes.RESEARCH DESIGN AND METHODSClinically diagnosed patients with type 2 diabetes (n = 258; diabetes duration: 0.01–31 years) were evaluated using a new biomarker detecting autoantibodies directed to the extracellular domain of the neuroendocrine autoantigen IA-2 (IA-2ec). The proportion of IA-2ec autoantibodies was also evaluated in newly diagnosed patients with type 1 diabetes (n = 150; diabetes duration: 0.04–0.49 years). In addition, IA-2 (intracellular domain), GAD65, and zinc transporter 8 autoantibodies were assayed.RESULTSIA-2ec autoantibodies were detected in patients with type 1 diabetes and, surprisingly, in 5% of patients with type 2 diabetes without serologic responses to other IA-2 antigenic epitopes or other islet autoantigens. We also assessed the ability of IA-2ec–derived peptides to elicit CD4+ T-cell responses by stimulating peripheral blood mononuclear cells from patients with type 1 diabetes (n = 18) and HLA-matched healthy subjects (n = 13) with peptides and staining with the peptide/DQ8-specific tetramers, observing disease-associated responses to previously unreported epitopes within IA-2ec.CONCLUSIONSWe developed a new antibody biomarker identifying novel antigenic determinants within the N terminus of IA-2. IA-2ec autoantibodies can be detected in patients with type 1 diabetes and in a subgroup of adult autoimmune patients with type 2 diabetes phenotype negative for conventional islet autoantibody testing. These observations suggest that islet autoimmunity may be more common in clinically diagnosed type 2 diabetes than previously observed.

Islet cell autoantigen of 69 kDa (ICA69) is a cytosolic protein of still unknown function. Involvement of ICA69 in neurosecretion has been suggested by the impairment of acetylcholine release at neuromuscular junctions upon mutation of its homologue gene ric-19 in C. elegans. In this study, we have further investigated the localization of ICA69 in neurons and insulinoma INS-1 cells. ICA69 was enriched in the perinuclear region, whereas it did not co-localize with markers of synaptic vesicles/synaptic-like microvesicles. Confocal microscopy and subcellular fractionation in INS-1 cells showed co-localization of ICA69 with markers of the Golgi complex and, to a minor extent, with immature insulin-containing secretory granules. The association of ICA69 with these organelles was confirmed by immunoelectron microscopy. Virtually no ICA69 immunogold labeling was observed on secretory granules near the plasma membrane, suggesting that ICA69 dissociates from secretory granule membranes during their maturation. In silico sequence and structural analyses revealed that the N-terminal region of ICA69 is similar to the region of arfaptins that interacts with ARF1, a small GTPase involved in vesicle budding at the Golgi complex and immature secretory granules. ICA69 is therefore a novel arfaptin-related protein that is likely to play a role in membrane trafficking at the Golgi complex and immature secretory granules in neurosecretory cells.

Islet cell autoantigen 69-kDa (ICA69), protein product of the human ICA1 gene, is one target of the immune processes defining the pathogenesis of Type 1 diabetes. We have characterized the genomic structure and functional promoters within the 5-regulatory region of ICA1. 5-RNA ligase-mediated rapid amplification of cDNA ends evaluation of ICA1 transcripts expressed in human islets, testis, heart, and cultured neuroblastoma cells reveals that three 5-untranslated region exons are variably expressed from the ICA1 gene in a tissue-specific manner. Surrounding the transcription initiation sites are motifs characteristic of non-TATA, non-CAAT, GC-rich promoters, including consensus Sp1/GC boxes, an initiator element, cAMP-responsive element-binding protein (CREB) sites, and clusters of other putative transcription factor sites within a genomic CpG island. Luciferase reporter constructs demonstrate that the first two ICA1 exon promoters reciprocally stimulate luciferase expression within islet-(RIN 1046-38 cells) and brain-derived (NMB7) cells in culture; the exon A promoter exhibits greater activity in islet cells, whereas the exon B promoter more efficiently activates transcription in neuronal cells. Mutation of a CREB site within the ICA1 exon B promoter significantly enhances transcriptional activity in both cell lines. Our basic understanding of expression from the functional core promoter elements of ICA1 is an important advance that will not only add to our knowledge of the ICA69 autoantigen but will also facilitate a rational approach to discover the function of ICA69 and to identify relevant ICA1 promoter polymorphisms and their potential associations with disease.