Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0011849 (diabetes)
 277,896 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glutamic acid decarboxylase (GAD) has been shown to be a target of autoantibodies in insulin-dependent diabetes (IDD). Two forms of GAD, with molecular weights of 67,000 and 65,000, have been cloned from separate genes. As pancreatic islet beta cell destruction DD is an autoimmune process mediated by T cells, we sought to determine if recombinant GAD67 was recognized by T cells in IDD subjects and particularly their first-degree relatives with islet cell antibodies known to be at risk for IDD. The central regions of human islet and brain GAD67 (amino acids 208-404) were cloned as fusion proteins with glutathione-S-transferase (GST). Proliferation of peripheral blood T cells in the presence of recombinant GAD67 was significantly higher in both at-risk relatives and recent-onset IDD subjects than in other autoimmune disease subjects and human histocompatibility leukocyte antigen (HLA)-matched healthy controls. Thus, 12 of 29 (41%) at-risk relatives and 11 of 29 (38%) recent-onset IDD subjects responded to GAD67, compared with 1 of 7 (14%) other autoimmune disease subjects and 1 of 23 (4%) HLA-matched controls. T cell responses to GST alone or to tetanus toxoid were not different between the groups. These findings demonstrate that GAD67 is a target autoantigen of T cells in IDD and suggest the possibility that GAD-reactive T cells may delineate asymptomatic subjects at increased risk for IDD.
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PMID:Glutamic acid decarboxylase 67-reactive T cells: a marker of insulin-dependent diabetes. 842 22

The GABA synthesizing enzyme GAD is a prominent islet cell autoantigen in type I diabetes. The two forms of GAD (GAD64 and GAD67) are encoded by different genes in both rats and humans. By in situ hybridization analysis of rat and human pancreases, expression of both genes was detected in rat islets, whereas only GAD64 mRNA was detected in human islets. Immunocytochemical analysis of rat and human pancreatic sections or isolated islets with antibodies to GAD64 and GAD67 in combination with antibodies to insulin, glucagon, or SRIF confirmed that a GAD64 and GAD67 expression were beta-cell specific in rat islets. In contrast, only GAD64 was detected in human islets and was, in addition to beta-cells, also surprisingly localized to some alpha-cells, delta-cells, and PP-cells. In long-term (4 wk) monolayer cultures of newborn rat islet cells, GAD64 expression remained beta-cell specific as observed in vivo, whereas GAD67 was localized not only to the beta-cells but also in the alpha-cells and delta-cells. A small but distinct fraction of GAD positive cells in these monolayer cultures did not accumulate GABA immunoreactivity, which may indicate cellular heterogeneity with respect to GABA catabolism or GAD enzyme activity. In a rat insulinoma cell line (NHI-6F) producing both glucagon and insulin depending on the culture conditions, GAD64 expression was detected only in cultures in which the insulin producing phenotype dominated. In conclusion, these data demonstrate that the two GAD isoforms are differentially expressed in rat and human islets but also that the expression differs according to culture conditions. These findings emphasize the need to consider both the species and culture conditions of islets.
Diabetes 1993 Mar
PMID:Differential expression of glutamic acid decarboxylase in rat and human islets. 843 19

Glutamic acid decarboxylase autoantibodies may aid in rapid screening strategies predicting IDDM before clinical onset. Rat islets contain GAD65 and GAD67 autoantibody targets, but human islets express only GAD65, now confirmed by direct immunoprecipitation from radiolabeled rat and human islets. Because human IDDM involves beta-cell-specific autoimmunity, we tested 190 new IDDM patients and 51 healthy control subjects for antibodies to recombinant human islet GAD65, rat islet GAD67, or human insulinoma/cerebellum GAD67, each expressed separately in hamster fibroblasts. By using immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and densitometric fluorogram scanning, 132 of 190 (70%) of new IDDM patients had GAD65 autoantibodies, whereas only 17 of 190 (9%) had antibodies to rat GAD67 (P < 0.001). Of healthy control subjects, 2 of 51 (3.9%) and 1 of 51 (1.9%) had antibodies to GAD65 and GAD67, respectively. All 17 GAD67 antibody-positive patients also had GAD65 antibodies; 14 of 17 with greater GAD65 than GAD67 index. Control studies showed comparable reactivity between recombinant rat and human GAD67 and between different subcellular preparations of recombinant GAD67 of either species. In conclusion, only GAD65 is expressed in human islets, the autoantibody response is primarily to this isoform, and GAD67 antibodies add little to IDDM detection.
Diabetes 1993 Apr
PMID:Autoantibodies in IDDM primarily recognize the 65,000-M(r) rather than the 67,000-M(r) isoform of glutamic acid decarboxylase. 845 15

Glutamic acid decarboxylase (GAD) is the enzyme responsible for synthesis of the neurotransmitter gamma-aminobutyric acid in neurons and pancreatic beta cells. It is represented by two isoforms, GAD-65 and GAD-67, which are the products of two different genes and differ substantially only at their N-terminal regions. GAD-65 is a dominant autoantigen in stiff-man syndrome and insulin-dependent diabetes mellitus. In neurons and beta cells, GAD is concentrated around synaptic vesicles and synaptic-like microvesicles, respectively, as well as in the area of the Golgi complex. The mechanisms responsible for specific targeting of GAD to these organelles are not yet understood. The elucidation of the mechanism of subcellular targeting of GAD may be relevant to understanding its role as an autoantigen. In this study, the cloned genes for GAD-65 and GAD-67 were expressed separately in Chinese hamster ovary (CHO) cells and COS cells. While GAD-67 had a diffuse cytoplasmic localization, GAD-65 had a punctate distribution, with most of the immunoreactivity being concentrated in the area of the Golgi complex. A chimeric protein in which the 88 N-terminal amino acids of GAD-67 were replaced by the 83 N-terminal amino acids of GAD-65 was targeted to the Golgi complex, indicating that the N-terminal region of GAD-65 contains a targeting signal sufficient for directing the remaining portion of the molecule, highly similar in GAD-65 and GAD-67, to the Golgi complex-associated structures.
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PMID:Association of GAD-65, but not of GAD-67, with the Golgi complex of transfected Chinese hamster ovary cells mediated by the N-terminal region. 846 26

Plasmids containing cDNA for the rat 67- and 65-kD isoforms of glutamate decarboxylase (GAD-67 and GAD-65) were expressed in COS-cells, and lysates of [35S]methionine-labeled cells were used for immunoprecipitations. Sera from 38 patients with type 1 (insulin-dependent) diabetes mellitus, which precipitated a 64-kD antigen from rat islets, reacted with recombinant GAD-65 in relation to their anti-64-kD titers. The eight strongest sera also precipitated recombinant GAD-67, suggesting that certain epitopes are common to both isoforms. Subsequently, [35S]methionine-labeled GAD-65 was purified from COS cell lysates and employed in a binding assay with 50 sera of patients with recent onset of type 1 diabetes mellitus. 38 sera (76%) precipitated labeled GAD-65 with titers that correlated with islet cell antibodies (ICA), determined in a standard immunofluorescence assay. 2 sera were GAD positive but ICA negative, 4 were positive only for ICA, and 6 were negative for both GAD and ICA, as were the sera of 20 controls. The data illustrate that antibodies against GAD-65 are present in a majority of patients with type 1 diabetes mellitus and that autoantibodies against other islet cell antigens also exist. The radioligand-binding assay, which is convenient and sensitive for detecting GAD antibodies, will facilitate the screening of individuals with autoimmune islet cell disease.
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PMID:Demonstration of GAD-65 as the main immunogenic isoform of glutamate decarboxylase in type 1 diabetes and determination of autoantibodies using a radioligand produced by eukaryotic expression. 848 75

Macaca nemestrina, which may have larger and more numerous pancreatic islets than other species, was used for large scale islet isolation by ductal collagenase perfusion and Ficoll gradient centrifugation. The average yield was 51,000 islet equivalents per pancreas, or 8,750 islets equivalents per g. The average purity was 91%, often exceeding 95%. These are the highest reported size, purity, and yield per g of any nonautomated primate islet series. Perifusion with glucose, arginine, and isobutylmethylxanthine showed appropriate biphasic insulin secretion. Unlike that in the rat, human islet glutamic acid decarboxylase (GAD) isoform expression is restricted. However, glycemic regulation of GAD expression has been shown only in rats. We, therefore, tested hypotheses that M. nemestrina islets also have restricted GAD expression, that GAD expression in primates is stimulated by glucose, and that this stimulation remains restricted to the 64,000 mol wt (GAD65) isoform. Immunoprecipitation of labeled islet extracts showed that GAD65 expression increased 16.7 +/- 0.6-fold during high glucose in vitro culture. After controlling for observed increases in protein synthesis, specific glucose stimulation was still 4.2 +/- 0.2-fold. Specific antisera revealed no GAD67 expression under basal conditions, and isoform restriction was maintained during stimulation. Increased GAD65 synthesis thus accounts for glucose stimulation of 64K expression. These time- and concentration-dependent effects of glucose suggest that hyperglycemia increases autoantigenicity and may accelerate beta-cell destruction in primates, supporting a role for beta-cell rest in insulin-dependent diabetes mellitus prevention.
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PMID:Regulation of glutamic acid decarboxylase diabetes autoantigen expression in highly purified isolated islets from Macaca nemestrina. 850 67

Glutamic acid decarboxylase (GAD) is a pancreatic islet autoantigen in insulin-dependent diabetes (IDD). Two forms of GAD, GAD65 and GAD67, have been identified in brain but human islets have been reported to express only GAD65. We have isolated full length GAD67 cDNA by polymerase chain reaction (PCR) cloning from human pancreas. Sequence analysis reveals only four nucleotide differences between human pancreas and brain GAD67, two of which result in amino acid changes. These differences are probably individual-specific and reflect allelic variation. Using mRNA from isolated human islets a partial sequence was obtained by PCR cloning that was identical to the midregion of pancreas GAD67 cDNA. GAD67 has previously been shown to be a target of both autoantibodies and autoreactive T cells in IDD. The presence of GAD67 in human pancreas implies that this form of GAD, as well as GAD65, has a pathogenic role in IDD.
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PMID:Molecular cloning of full-length glutamic acid decarboxylase 67 from human pancreas and islets. 850 2

Glutamic acid decarboxylase (GAD) catalyzes formation of gamma-aminobutyric acid from glutamic acid and is a major autoantigen in insulin-dependent diabetes mellitus. Its two isoforms, GAD65 and GAD67, are encoded by two separate genes. We prepared human islet cDNA library and screened it with cDNA probes of rat brain GAD67. We cloned the cDNA for GAD67, the large isoform of glutamic acid decarboxylase, and determined its nucleotide sequence. Sequencing of the resulting clone identified a 1,785 residue open-reading frame encoded a 594 amino acid polypeptide that showed a 99.4% similarity with GAD67 from human brain. The bacterially expressed human islet GAD67 protein was enzymatically active and immunoreactive. The isolation of cDNA for this additional islet GAD isoforms will be important in studying the etiology and pathogenesis of IDDM.
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PMID:Cloning and expression of large isoform of glutamic acid decarboxylase from human pancreatic islet. 850 3

One of the major beta-cell autoantigens associated with IDDM is GAD. Although GAD expression has been detected in adult islets, transcriptional expression of the GAD genes has not been reported during human pancreatic ontogeny. We therefore analyzed patterns of GAD gene transcription by quantitating the mRNAs encoding both the 65- and 67-kDa isoforms (GAD65 and GAD67, respectively) in human fetal, postnatal, and adult pancreases, as well as in isolated adult islets, and examined their tissue-specific expression. Significant levels of pancreatic GAD65 transcripts were already detected at 13 weeks of gestation and were expressed at higher levels in the fetal and infantile pancreas than in the adult pancreas. Isolated adult pancreatic islets were highly enriched in GAD65 mRNA. In contrast, GAD67 transcripts were not detectable in fetal and postnatal pancreases. In addition to the pancreas, marked GAD expression was detected in the brain, whereas other tissues examined contained either low or undetectable GAD transcripts. Triple immunofluorescent staining of fetal and adult pancreases revealed colocalization of GAD65 with alpha- and beta-cells. In the fetal pancreas, strong immunoreactivity for GAD65 was also evident in epithelial cells, which lacked expression of insulin or glucagon, some of which were present in the ductal epithelium, suggesting that GAD65 expression might correlate with endocrine determination. In summary, 1) this is the first demonstration of GAD65 expression in the human fetal pancreas, implicating a potential role during islet development, and 2) GAD65 may be a useful marker for the identification of primitive islet cells.
Diabetes 1996 Apr
PMID:Ontogeny and tissue distribution of human GAD expression. 860 72

To evaluate the association of autoimmunity to glutamic acid decarboxylase (GAD) with insulin-dependent diabetes mellitus (IDDM) and IDDM-associated human leukocyte antigen (HLA) types, we studied a unique group of 47 patients with autoimmune polyendocrine syndrome type 1, a recessive disease not associated with HLA. GAD65 antibodies (GAD65-Ab), GAD67-Ab, islet cell antibodies, and HLA-DQA1, -DQB1, and -DRB1 were analyzed in relation to IDDM or a decreased insulin secretory capacity. GAD65-Ab were found in six of the eight diabetic patients 0.9-8.0 yr before the onset of IDDM and in 16 (41%) nondiabetic patients during a follow-up of 2.4-19.5 yr. Eleven (28%) nondiabetic patients had GAD67-Ab and islet cell antibodies. Fasting C peptide (mean +/- SD, 0.5 +/- 0.24 vs. 1.03 +/- 0.49 nmol/L; P = 0.003) and first phase insulin response (75.6 +/- 37.9 vs. 166.4 +/- 112.7 mU/L; P = 0.019) were lower in patients with than in those without GAD65-Ab. No HLA genotype predominated in the IDDM patients or GAD65-Ab-positive nondiabetic patients, but the IDDM high risk genotypes were decreased in frequency among the patients with GAD65-Ab. In conclusion, nondiabetic autoimmune polyendocrine syndrome type 1 patients frequently have GAD65-Ab together with a decreased insulin secretory capacity, suggesting subclinical islet cell inflammation not invariably progressing to diabetes. This is not associated with HLA haplotypes conferring susceptibility to or protection from IDDM.
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PMID:Antibodies to glutamic acid decarboxylase and insulin-dependent diabetes in patients with autoimmune polyendocrine syndrome type I. 863 56


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