PARALLEL ACCELERATION OF PHOSPHOENOLPYRUVATE CARBOXYKINASE MESSENGER-RNA DEGRADATION AND INCREASE IN RIBONUCLEASE-ACTIVITY INDUCED BY INSULIN IN CULTURED RAT HEPATOCYTES

In cultured rat hepatocytes, glucagon increased phosphoen olpyruvate c arboxykinase mRNA transiently. Insulin, given at the maximal increase, enhanced the degradation by 3-fold. The levels of beta-actin mRNA and ribosomal RNA, which served as a control, remained unchanged. The tra nscriptional inhibitor, actinomycin D, or the serine/threonine phospha tase IIA inhibitor, okadaic acid, prevented the degradation of phospho enolpyruvate carboxykinase mRNA. This indicated that the degradation o f phosphoenolpyruvate carboxykinase mRNA requires the de novo synthesi s of a bona fide destabilizing factor and/or active protein phosphatas e. In vitro RNA degradation assays were developed in order to investig ate whether insulin-treated cells contained enhanced ribonuclease acti vity. Fractionated cytosolic extracts were prepared by removing cell o rganelles by differential centrifugation and thereafter part of the cy tosolic proteins by heat treatment. These extracts were incubated with exogenously added total RNA and the degradation of phosphoenolpyruvat e carboxykinase mRNA, beta-actin mRNA and 28S ribosomal RNA was studie d. In this assay, phosphoenolpyruvate carboxykinase mRNA and the other wise stable beta-actin mRNA and ribosomal RNA were degraded 3-fold fas ter by extracts from insulin-treated, than from untreated, cells. The increase in RNase activity induced by insulin could be prevented by tr eatment of cultured rat hepatocytes with actinomycin D, indicating tha t ongoing gene transcription was required. The 'in vivo' specificity o f the insulin effect on PCK mRNA degradation in cultured hepatocytes s eemed to be lost in the in vitro assay in cytosolic extracts due to th e disruption of the intracellular environment. Also in whole cell lysa tes, which were obtained by hypo-osmotic shock of the cells, and which contained the disrupted particulate and all soluble cellular componen ts, PCK mRNA as well as beta-actin mRNA and ribosomal RNA, was degrade d. The increase in ribonuclease activity due to insulin paralleled the insulin-induced acceleration of phosphoenolpyruvate carboxykinase mRN A degradation in cultured hepatocytes, which might indicate a function al correlation.