REGULATION OF GAD EXPRESSION IN ISLETS OF LANGERHANS OCCURS BOTH AT THE MESSENGER-RNA 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 immunoprecipit ations and analyses of enzyme activity, an increase in glutamate decar boxylase was detected in rat islets cultured at a glucose concentratio n 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 expressi on, 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 sig nificantly higher (82%, P < 0.005) content of glutamate decarboxylase mRNA in islets cultured at 11 mmol/l glucose. In parallel, human recom binant interleukin-1 beta, and diazoxide were tested for their effects on the expression of glutamate decarboxylase. Islets cultured at 11 m mol/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 cul tured at 11 mmol/l glucose alone, and similar decreases were noted on analysis of glutamate decarboxylase biosynthesis and mRNA. Islets cult ured at 11 mmol/l glucose in the presence of 22.5 mg/ml diazoxide exhi bited a significant reduction in enzyme activity (59%; P < 0.001) comp ared with those cultured at 11 mmol/l glucose only. This reduction, ho wever, was not accompanied by a decrease in the content of glutamate d ecarboxylase mRNA. In a separate set of experiments, the glutamate dec arboxylase mRNA content in human islets was determined by the polymera se chain reaction and was 27% higher in islets cultured at a high than at a low glucose concentration. In all experimental conditions, the r ate of insulin release followed the pattern of glutamate decarboxylase activity. These results suggest that the stimulatory or inhibitory ef fects of different agents on glutamate decarboxylase expression may be mediated both by a modulation of the glutamate decarboxylase mRNA con tent and by a modification at the protein level. Furthermore, they sug gest that the regulation of glutamate decarboxylase mRNA expression in the beta-cell co-varies with the expression of the insulin gene.