Parallel acceleration of phosphoenolpyruvate carboxykinase mRNA degradation and increase in ribonuclease activity induced by insulin in cultured rat hepatocytes - PubMed (original) (raw)
Parallel acceleration of phosphoenolpyruvate carboxykinase mRNA degradation and increase in ribonuclease activity induced by insulin in cultured rat hepatocytes
T Heise et al. Biol Chem. 1998 Jul.
Abstract
In cultured rat hepatocytes, glucagon increased phosphoenolpyruvate carboxykinase 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 transcriptional inhibitor, actinomycin D, or the serine/threonine phosphatase IIA inhibitor, okadaic acid, prevented the degradation of phosphoenolpyruvate carboxykinase mRNA. This indicated that the degradation of phosphoenolpyruvate carboxykinase mRNA requires the de novo synthesis of a bona fide destabilizing factor and/or active protein phosphatase. In vitro RNA degradation assays were developed in order to investigate whether insulin-treated cells contained enhanced ribonuclease activity. Fractionated cytosolic extracts were prepared by removing cell organelles by differential centrifugation and thereafter part of the cytosolic proteins by heat treatment. These extracts were incubated with exogenously added total RNA and the degradation of phosphoenolpyruvate carboxykinase mRNA, beta-actin mRNA and 28S ribosomal RNA was studied. In this assay, phosphoenolpyruvate carboxykinase mRNA and the otherwise stable beta-actin mRNA and ribosomal RNA were degraded 3-fold faster by extracts from insulin-treated, than from untreated, cells. The increase in RNase activity induced by insulin could be prevented by treatment of cultured rat hepatocytes with actinomycin D, indicating that ongoing gene transcription was required. The 'in vivo' specificity of the insulin effect on PCK mRNA degradation in cultured hepatocytes seemed to be lost in the in vitro assay in cytosolic extracts due to the disruption of the intracellular environment. Also in whole cell lysates, which were obtained by hypo-osmotic shock of the cells, and which contained the disrupted particulate and all soluble cellular components, PCK mRNA as well as beta-actin mRNA and ribosomal RNA, was degraded. The increase in ribonuclease activity due to insulin paralleled the insulin-induced acceleration of phosphoenolpyruvate carboxykinase mRNA degradation in cultured hepatocytes, which might indicate a functional correlation.
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