Mediators of Insulin-Dependent Messenger RNA Synthesis

Hargrove, James

Abstract

Insulin prevents the induction of mRNA for phosphoenolpyruvate carboxykinase by dubutyryl cyclic AMP and hydrocortisone in Reuber hepatoma cells but simultaneously acts in an additive fashion with these agents in inducing tyrosine aminotransferase. These results imply that these agents interact to promote or inhibit mRNA production from specific genes. Therefore, this project will test the idea that insulin, cyclic AMP and glucocorticoids alter production of specific messenger RNAs in Reuber hepatoma cells by altering the charge, abundance or intracellular distribution of genetic regulatory proteins. Two model proteins that translocate to the nucleus and are probably involved in transcriptional regulation (the glucocorticoid receptor and type II cyclic AMP-dependent protein kinase) will be identified in two-dimensional polyacrylamide gels (2D-PAGE) of radiolabeled extracts of whole cells, nuclei and chromatin. These proteins will be studied to find whether hormonal combinations that alter mRNA induction alter their activation or translocation to the nucleus. Activation will be sought as changes in the charge (phosphorylation state or other modification) of these receptors using 2D-PAGE. Translocation will be detected as decreased association of these proteins with the cytosol and increased association with the nucleus after hormonal stimulation, again identifying the receptors with 2D-PAGE. Cells will be treated with insulin to identify molecules whose charge or abundance is regulated by this hormone, and a molecule will be sought that becomes associated with chromatin after insulin treatment. Nuclear events such as translocation of regulatory molecules or altered phosphorylation of histones and other proteins will be identified and attempts will be made to correlate these events with altered production of messenger RNAs. Events that correlate with insulin's ability to abolish the induction of messenger RNA for phosphoenolpyruvate carboxykinase by dibutyryl cyclic AMP and glucocorticoids, will be pursued. If a putative regulator is found, I will attempt to purify it, study how it is activated, and determine how it associates with chromatin or DNA. Knowledge of how protein effectors are stimulated by insulin to regulate genetic events is fundamental to the understanding of long-term changes brought about by insulin, and conversely, of changes expected in the course of diabetes.