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.

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.

Structure of cat islet amyloid polypeptide and identification of amino acid residues of potential significance for islet amyloid formation.

Cats and humans, unlike most rodent species, develop amyloid in the islets of Langerhans in conjunction with non-insulin-dependent diabetes mellitus. The amyloid consists of a 37-amino acid polypeptide referred to as islet amyloid polypeptide (IAPP). The primary structures of IAPP from human and three rodent species have previously been determined. Sequence divergence was seen in the region corresponding to amino acid residues 20-29, which in human IAPP has been suggested to confer the amyloidogenic properties to the molecule. Using polymerase chain-reaction methodology, we determined the primary sequence of cat IAPP. Amino acid region 20-29 shows specific similarities and differences compared with human and rodent IAPP, respectively. A synthetic cat IAPP20-29 decapeptide formed amyloid fibrils spontaneously in vitro. Comparison between the structure and amyloid fibril-forming activity of various synthetic peptides suggests that the amino acid residues at positions 25-26 in mature IAPP are important for the amyloidogenic properties of the molecule.

The human islet amyloid polypeptide (IAPP) gene. Organization, chromosomal localization and functional identification of a promoter region.

We report the isolation and characterization of the human gene encoding islet amyloid polypeptide (IAPP). Previously characterized cDNA sequences correspond to three exons of which the first is noncoding. A functional promoter region was identified in the 5' flanking DNA; however, this was farther upstream than expected. Northern blot analysis of human insulinoma RNA revealed three IAPP mRNAs of sizes 1.2, 1.8 and 2.1 kb, in agreement with three polyadenylation signals present in the 3' end of the gene. In situ hybridization to metaphase chromosomes resulted in two distinct peaks on chromosome 12, at 12p12-p13 and 12q13-q14. Southern blot analysis of genomic DNA suggested a single IAPP locus but also indicated the presence of additional homologous sequences in human genomic DNA.

The transthyretin cDNA sequence is normal in transthyretin-derived senile systemic amyloidosis.

A variety of mutations leading to amino acid substitutions have been described in the transthyretin gene in association with different familial amyloidoses and have been implicated to be involved in the pathogenesis of amyloid deposits. However, there has been disagreement whether or not a transthyretin mutation is present in the most common form of transthyretin-derived amyloid, namely senile systemic amyloidosis. Therefore, the cDNA sequence of liver transthyretin was determined in a 91-year-old patient with typical senile systemic amyloidosis. This sequence was completely normal and lacked any variation. We conclude that in senile systemic amyloidosis factors other than the presence of a sequentially variant transthyretin must determine the amyloid fibril formation.

p-Chloroamphetamine releases both serotonin and dopamine into rat brain dialysates in vivo.

The effect of p-chloroamphetamine (PCA) on extracellular levels of endogenous 5-hydroxytryptamine (5-HT) and dopamine (DA) in the striatum and frontal cortex of the halothane-anesthetized rat was studied using an intracerebral dialysis method. PCA (5 mg/kg s.c.) induced an immediate, marked release of 5-HT into dialysates collected from the frontal cortex and striatum, an effect which lasted over 2 h. This treatment also caused a marked release of DA in the striatum. The results further emphasize the need to carefully assess the involvement of brain DA as well as 5-HT in PCA-induced behaviors.

Facial reactions to facial expressions in subjects high and low in public speaking fear.

This study investigated whether subjects high and low in public speaking fear react with different facial electromyographic (EMG) activities when exposed to negative and positive social stimuli. A High-fear and Low-fear group were selected by help of a questionnaire and were exposed to slides of angry and happy faces while facial-EMG from the corrugator and zygomatic muscle regions were measured. The subjects also rated the stimuli on different emotional dimensions. Consistent with earlier research it was found that Low fear subjects reacted with increased corrugator activity to angry faces and increased zygomatic activity to happy faces. The High fear group, on the other hand, did not distinguish between angry and happy faces. Rating data indicated that the High fear group perceived angry faces as being emotionally more negative. The present results are consistent with earlier studies, indicating that the facial-EMG technique is sensitive to detect differential responding among clinical interesting groups, such as people suffering from social fears.

Islet amyloid polypeptide stimulates cyclic AMP accumulation via the porcine calcitonin receptor.

CHO-cells stably transfected with an expression vector for the porcine calcitonin receptor were exposed to various concentrations of IAPP, CGRP or calcitonin from different species. In these, but not in untransfected cells, rat IAPP mediated cAMP accumulation at concentrations above 25 nM. This potency was three orders of magnitude lower than that of porcine calcitonin and two and four orders of magnitude lower than those of human and salmon calcitonin, respectively. Human beta-CGRP had an effect similar to rat IAPP whereas human alpha-CGRP was at least one order of magnitude less potent than rat IAPP. COS cells expressing recombinant porcine calcitonin receptors transiently or stably showed a different pattern of responses to calcitonin, IAPP and the CGRPs. In these cells, rat IAPP was as potent an inducer of cAMP as was salmon or porcine calcitonin and more potent than human calcitonin or the CGRPs. The dissociation constants for salmon calcitonin binding to the porcine calcitonin receptors on CHO and COS cells were 0.2 nM and 2.0 nM and the corresponding number of binding sites per cell were approximately 7 x 10(4) and 2 x 10(6), respectively. These results demonstrate that IAPP and CGRP can mediate signal transduction via the porcine calcitonin receptor and provide a possible explanation for the calcitonin-like effects of pharmacological levels of IAPP and CGRP administrated in vivo. The difference between CHO and COS cells in their relative response to the various ligands may relate to the difference in receptor number, post-transcriptional processing, or to dissimilarities in the signal transduction pathways between the two cell types.

Islet amyloid polypeptide--hen or egg in type 2 diabetes pathogenesis?

Islet amyloid polypeptide (IAPP or amylin) was first identified as the major peptide constituent of amyloid deposited in the islets of Langerhans in patients with type-2 diabetes mellitus or in insulinomas. It was subsequently shown that IAPP is produced by the pancreatic beta-cells, co-stored and co-released with insulin. IAPP is homologous with the neuropeptide calcitonin gene-related peptide (CGRP) and has therefore been assumed to have a function as an endocrine, paracrine or autocrine hormone. This has prompted the search for its physiological function as well as a putative pathogenic role in type 2 diabetes mellitus.

Islet amyloid polypeptide in the rabbit and European hare: studies on its relationship to amyloidogenesis.

In this study, we determined the cDNA-predicted amino acid sequence of positions 9-31 of islet amyloid polypeptide from the rabbit and European hare. A synthetic rabbit/hare islet amyloid polypeptide 20-29 peptide was subsequently shown to be strongly fibrillogenic in vitro even though the putative amyloidogenic AILS sequence at positions 25-28 of human and cat islet amyloid polypeptide is modified in the rabbit and hare by a substitution of phenylalanine for leucine at position 27 (i.e. AIFS). Although islet amyloid polypeptide of both the rabbit and hare has an amyloidogenic sequence and is in fact amyloidogenic in vitro, the apparent lack of in vivo islet amyloidosis in rabbits and hares may be related to relatively low levels of islet amyloid polypeptide production by the islet beta cells in these species. This was supported by our findings that there is no substantial immunoreactivity in either rabbit or hare islets, and no measurable amount either in extracts of rabbit pancreases, or in rabbit plasma. This study supports the need for at least two prerequisites for the development of islet amyloidosis in vivo: an inherent fibrillogenic sequence within the islet amyloid polypeptide molecule and an adequate local concentration of islet amyloid polypeptide to promote self aggregation and formation of islet amyloid.

Islet amyloid polypeptide: demonstration of mRNA in human pancreatic islets by in situ hybridization in islets with and without amyloid deposits.

Islet amyloid polypeptide which is normally coexpressed with insulin in beta cells, forms amyloid deposits especially in islets of Type 2 (non-insulin-dependent) diabetic subjects. Occurrence of islet amyloid is paradoxically associated with loss of islet amyloid polypeptide immunoreactivity in beta cells. The present study was undertaken to examine whether the islet amyloid polypeptide gene is expressed in islets with decreased islet amyloid polypeptide immunoreactivity. Pancreatic tissue from 14 patients, 7 with Type 2 diabetes and 7 non-diabetic, were obtained at autopsy or surgery and studied for islet amyloid polypeptide expression by in situ hybridization and for presence of insulin and islet amyloid polypeptide by immunohistochemistry. Six of the specimens from the diabetic and three of those from the non-diabetic patients had varying degrees of islet amyloid polypeptide-derived islet amyloid. Amyloid deposits were associated with decreased numbers of beta cells with islet amyloid polypeptide immunoreactivity despite an apparent normal frequency of insulin-containing cells. This discrepancy might reflect an alteration in islet amyloid polypeptide production or processing at a transcriptional or post-transcriptional level. In contrast to the varying immunohistochemical patterns, islets of all categories showed strong labelling using an islet amyloid polypeptide probe for in situ hybridization. It is concluded that islet amyloid polypeptide production is not altered at the transcriptional level. The following possibilities remain: (1) islet amyloid polypeptide production may be altered at a post-transcriptional level or (2) that islet amyloid polypeptide production is normal but the reduced immunoreactivity of the cells reflects a reduced storage of IAPP in secretory granules.(ABSTRACT TRUNCATED AT 250 WORDS)

Islet amyloid polypeptide in patients with pancreatic cancer and diabetes.

BACKGROUND: The diabetes mellitus that occurs in patients with pancreatic cancer is characterized by marked insulin resistance that declines after tumor resection. Islet amyloid polypeptide (IAPP), a hormonal factor secreted from the pancreatic beta cells, reduces insulin sensitivity in vivo and glycogen synthesis in vitro. In this study, we examined the relation between IAPP and diabetes in patients with pancreatic cancer. METHODS: We measured IAPP in plasma from 30 patients with pancreatic cancer, 46 patients with other cancers, 23 patients with diabetes, and 25 normal subjects. IAPP immunoreactivity and IAPP messenger RNA were studied in pancreatic cancers, pancreatic tissue adjacent to cancers, and normal pancreatic tissue. RESULTS: Plasma IAPP concentrations were elevated in the patients with pancreatic cancer as compared with the normal subjects (mean [+/- SD], 22.3 +/- 13.6 vs. 8.0 +/- 5.0 pmol per liter; P < 0.001), normal in the patients with other cancers, and normal or low in the patients with diabetes. Among the patients with pancreatic cancer, the concentrations were 25.0 +/- 8.7 pmol per liter in the 7 patients with diabetes who required insulin, 31.4 +/- 12.6 pmol per liter in the 11 patients with diabetes who did not require insulin, and 12.2 +/- 2.4 pmol per liter in the 9 patients with normal glucose tolerance (3 patients had impaired glucose tolerance; their mean plasma IAPP concentration was 11.7 +/- 5.5 pmol per liter). Plasma IAPP concentrations decreased after surgery in the seven patients with pancreatic cancer who were studied before and after subtotal pancreatectomy (28.9 +/- 16.4 vs. 5.6 +/- 3.4 pmol per liter, P = 0.01). Pancreatic cancers contained IAPP, but the concentrations were lower than in normal pancreatic tissue (17 +/- 16 vs. 183 +/- 129 pmol per gram, P < 0.001). In samples from the patients with both pancreatic cancer and diabetes, immunostaining for IAPP was reduced in islets of pancreatic tissue surrounding the tumor; in situ hybridization studies suggested that transcription occurred normally in these islets. CONCLUSIONS: Plasma IAPP concentrations are elevated in patients with pancreatic cancer who have diabetes. Since IAPP may cause insulin resistance, its overproduction may contribute to the diabetes that occurs in these patients.