F. A. Karlsson

Expression of the 64 kDa/glutamic acid decarboxylase rat islet cell autoantigen is influenced by the rate of insulin secretion

SummaryThis study examined the relationship between insulin secretion and expression of the 64 kDa/glutamic acid decarboxylase autoantigen in pancreatic islets. Islets isolated from Wistar rats were cultured for 3 days under different conditions: in 5.5 mmol/l glucose with or without a-ketoisocaproic acid or glipizide and in 28 mmol/l glucose with or without diazoxide. The 64 kDa/glutamic acid decarboxylase autoantigen was precipitated from lysates of [35S]-methionine-labelled islets with sera from patients with Type 1 (insulin-dependent) diabetes mellitus and identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and fluorography. In parallel, insulin contents of the islets and the media were determined as well as the rates of glucose-stimulated (pro)insulin biosynthesis. α-Ketoisocaproic acid and glipi zide were found to stimulate the expression of the 64 kDa/glutamic acid decarboxylase autoantigen and also the rate of insulin secretion. Diazoxide on the other hand reduced the rate of the 64 kDa/glutamic acid decarboxylase autoantigen synthesis in parallel with an inhibition of glucose-stimulated insulin release. Under most of the conditions employed, (pro)insulin biosynthesis was not affected. The correlation found between the rate of insulin release and expression of the 64 kDa/glutamic acid decarboxylase auto-antigen might provide an explanation for the earlier observed relationship between the functional demands on the Beta cells and their rate of destruction which may result in diabetes.

Human autoantibodies react with glutamic acid decarboxylase antigen in human and rat but not in mouse pancreatic islets.

SummaryThe presence of one of the major targets for auto-antibodies in Type 1 (insulin-dependent) diabetes mellitus, the enzyme glutamic acid decarboxylase, was studied in human, rat and mouse pancreatic tissue using immunoprecipitation and immunohistochemical techniques. Immunoprecipitation of glutamic acid decarboxylase was attempted with lysates of [35S]-methionine-labelled rat or mouse pancreatic islets using two different glutamic acid decarboxylase antisera, one mouse monoclonal antibody raised against the 65 kDa isoform of the enzyme, sera from six patients with Type 1 diabetes, one patient with stiff-man syndrome and sera from 19 non-obese diabetic mice. The same sera were used for immunoperoxidase staining of cryosections of human, rat or mouse pancreas. Using patient sera glutamic acid decarboxylase was detected by immunoprecipitations from isolated rat islets but not from islets of five different mouse strains tested, including the non-obese diabetic mouse. When using the non-obese diabetic mouse sera, glutamic acid decarboxylase could not be detected in either rat or mouse tissue. Immunoperoxidase staining demonstrated high levels of glutamic acid decarboxylase in human and rat pancreatic islets but low levels in mouse islets. Direct measurements of enzyme activity showed glutamic acid decarboxylase to be present in mouse islets at a level of about 40% of that in rat islets, and subsequent Western blot analyses indicated that mouse islets express the 67 kDa isoform, whereas in rat islets both the 67 and 65 kDa isoforms are present. The species difference at the level of one of the major islet cell autoantigens in Type 1 diabetes thus indicates that human or rat and mouse islets, respectively, express immunologically distinct forms of glutamic acid decarboxylase and differ in their way of regulating the enzyme production.

Autoantibodies to epithelial cells in patients on long-term therapy with leucocyte-derived interferon-alpha (IFN-α)

During routine screening for anti‐nuclear antibodies, using rat liver tissue as substrate, a reactivity against bile duct epithelium was observed in sera from carcinoid tumour patients given human leucocyte‐derived IFN‐a (HuLe IFN‐a). In a retrospective study, initiated by this observation, the development of serum antibodies to bile duct epithelium was observed in nine out of 12 patients with carcinoid tumours and in three out of 14 patients with hairy‐cell leukaemia during their treatment with HuLe IFN‐α. However, no bile duct reactivity was observed in sera from carcinoid or hairy‐cell leukaemia in patients given recombinant IFN‐α. When analysing the reactivity of positive sera against a panel of rat and human tissues, a uniform reactivity was observed against simple epithelial cells lining the gastrointestinal tract, pancreatic secretory ducts, fallopian tube, kidney tubuli. mesothelium and also against carcinoid tumour cells. The mechanisms promoting autoreactivity against this simple epithelial cell auloantigen is so far unknown. The cytoplasmic as well as the restricted staining pattern of simple epithelial cells may indicate autoreactivity against certain cytoskelelal intermediate filaments, such as cytokeratin19, 18 and8, known to be exclusively present in simple epithelial cells and tumours derived from them.

Absence of autoantibodies against glutamate decarboxylase (GAD) in the non-obese diabetic (NOD) mouse and low expression of the enzyme in mouse islets

GAD is a major islet cell autoantigen in human type 1 diabetes mellitus. Autoantibodies are preferentially directed against the 65‐kD isoform of the enzyme which is the only form expressed in human islets of Langerhans. The NOD mouse is a spontaneous model of type I diabetes, frequently employed in studies dealing with the immunopathogenesis of the disease. In the present study the reactivity of sera from 34 prediabetic and 15 diabetic NOD mice was tested against GAD protein present in islets of Langerhans and cerebellum, and against recombinant., semi‐purified GAD‐65 and GAD‐67. A rabbit antiserum (K2) raised against GAD‐67 could readily recognize the recombinant GAD‐67 and the isoform present in rat and mouse islets and mouse brain. A MoAb (GAD‐6) specific for the GAD‐65 isoform reacted against the recombinant GAD‐65 and the isoform present in rat islets and mouse brain, whereas no reactivity was observed when using mouse islets. However, when testing the NOD mice sera by immunohistochemistry, immunoprecipitation and Western blot, no reactivity against any of the isoforms of GAD could be detected. Using reverse transcription polymerase chain reaction (PCR), GAD‐67 mRNA could be detected in mouse and rat islets and in mouse brain. GAD‐65 mRNA could also be detected in rat islets and mouse brain, but apparently a much lower copy number is present in mouse islets. These findings stress important diabetes in the immune response occurring in the animal model NOD mouse compared with human type 1 diabetes, and emphasize that human and animal type 1 diabetes possibly represent the final outcome of several different etiological factors.

Regulation of GAD expression in islets of Langerhans occurs both at the mRNA and protein level.

The expression of the autoantigen glutamate decarboxylase in islets of Langerhans was investigated under different culture conditions, which affect the functional activity of the beta-cell. Using immunoprecipitations and analyses of enzyme activity, an increase in glutamate decarboxylase was detected in rat islets cultured at a glucose concentration of 11 mmol/l compared with those cultured at 5.6 mmol/l glucose. To determine whether the change was induced at the level of mRNA expression, total RNA was extracted from rat islets cultured at 5.6 or 11 mmol/l glucose, reverse transcribed and amplified by the polymerase chain reaction. Comparative quantitation in a phosphor imager revealed a significantly higher (82%, P < 0.005) content of glutamate decarboxylase mRNA in islets cultured at 11 mmol/l glucose. In parallel, human recombinant interleukin-1 beta, and diazoxide were tested for their effects on the expression of glutamate decarboxylase. Islets cultured at 11 mmol/l glucose in the presence of 40 U/ml of interleukin-1 beta, showed a 63% decrease (P < 0.005) in enzyme activity compared with those cultured at 11 mmol/l glucose alone, and similar decreases were noted on analysis of glutamate decarboxylase biosynthesis and mRNA. Islets cultured at 11 mmol/l glucose in the presence of 22.5 mg/ml diazoxide exhibited a significant reduction in enzyme activity (59%; P < 0.001) compared with those cultured at 11 mmol/l glucose only. This reduction, however, was not accompanied by a decrease in the content of glutamate decarboxylase mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoimmune endocrinopathies 5 Autoimmune disease of the adrenal cortex, pituitary, parathyroid glands and gastric mucosa

Over the past several years, our laboratory has been interested in characterizing autoantigens in the adrenal cortex and the stomach. In this paper, we discuss in some detail the current knowledge in this field, We also review observations made by other investigators of autoimmune diseases of the pituitary and parathyroid glands. As can be seen, the presumed immunological target structures in these latter organs remain to be elucidated.

Autoantibodies against adrenal medulla in type 1 and type 2 diabetes mellitus: no evidence for an association with autonomic neuropathy

Objective. To examine the role of autoimmunity in the development of autonomic neuropathy in diabetes mellitus.