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

Abstract

The expression of the autoantigen glutamate decarboxylase in islets of Langerhans was investigated under different culture conditions, which affect the functional activity of the β-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/1 compared with those cultured at 5.6 mmol/1 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/1 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/1 glucose. In parallel, human recombinant interleukin-lβ, and diazoxide were tested for their effects on the expression of glutamate decarboxylase. Islets cultured at 11 mmol/1 glucose in the presence of 40 U/ml of interleukin-1β, showed a 63% decrease ( P < 0.005) in enzyme activity compared with those cultured at 11 mmol/1 glucose alone, and similar decreases were noted on analysis of glutamate decarboxylase biosynthesis and mRNA. Islets cultured at 11 mmol/1 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/1 glucose only. This reduction, however, was not accompanied by a decrease in the content of glutamate decarboxylase mRNA. In a separate set of experiments, the glutamate decarboxylase mRNA content in human islets was determined by the polymerase chain reaction and was 27% higher in islets cultured at a high than at a low glucose concentration. In all experimental conditions, the rate of insulin release followed the pattern of glutamate decarboxylase activity. These results suggest that the stimulatory or inhibitory effects of different agents on glutamate decarboxylase expression may be mediated both by a modulation of the glutamate decarboxylase mRNA content and by a modification at the protein level. Furthermore, they suggest that the regulation of glutamate decarboxylase mRNA expression in the β-cell co-varies with the expression of the insulin gene.