Regulation of Tyrosine Aminotransferase by Insulin and Cyclic AMP: Similar Effects on Activity but Opposite Effects on Transcription

Abstract

Tyrosine aminotansferase (TAT) is a liver-specific enzyme whose activity is subject to positive regulation by several agents including insulin and agonists that increase the intracellular concentration to cAMP. To further characterize the mechanism of insulin action and the interaction between cAMP and insulin several types of experiments were performed in a rat hepatoma cell-line. In the presence of the transcriptional inhibitor 5,6-dichloro-1-.beta.-D-ribofuranoyslbenzimidazole, TAT enzyme activity remains inducible by insulin and to a lesser extent by the cAMP analog (Bu)2cAMP. This suggests that transcriptional events are not necessary for the insulin-mediated increase in TAT activity, and also suggests a dualistic mechanism for the cAMP-induced increase in TAT activity. Surprisingly, using a cDNA probe for mRNATAT, it was found that insulin causes a decrease in hybridizable mRNATAT, in addition to causing a partial inhibition of the increase of hybridizable TAT transcript caused by (Bu)2cAMP. Examination of the rate of transcription of the TAT gene by a nuclear run-off assay shows that insulin causes a decrease in the transcription of the TAT gene by greater than 50%, which is sufficient to account for the decrease in hybridizable mRNATAT. As expected (Bu)2cAMP increases the trascription of TAT, but combined with insulin a complete inhibition of the increase in TAT transcription caused by (Bu)2cAMP is observed. To address the possibility that insulin acts posttranslationally to increase TAT activity, the t1/2 of TAT protein was measured in the presence and absence of insulin. It was found that insulin causes a decrease in the rate of total cellular protein turnover by 35%, and increases the t1/2 of TAT protein by 55%. In addition, insulin caused an increase in the rate of synthesis of the TAT protein. This increase in the t1/2 of TAT caused by insulin coupled with the increased rate of synthesis is sufficient to account for the increase in TAT activity even though the amount of transcript is diminished. Thus, insulin has transcriptional, translational, and posttranslational effects on TAT, the latter two being responsible for the increase in TAT activity.