The glucagon‐insulin antagonism in the regulation of cytosolic protein binding to the 3′ end of phosphoenolpyruvate carboxykinase mRNA in cultured rat hepatocytes Possible involvement in the stabilization of the mRNA

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Abstract
Since protein binding to the 3′ end of mRNA is believed to be involved in the control of mRNA stability, the time course of alterations in glucagon‐induced phosphoenolpyruvate‐carboxykinase‐mRNA (PCK) levels, in the absence and presence of insulin, was correlated with the time course of changes in the binding of cytosolic protein from 24‐h cultured rat hepatocytes to the 3′ end of PCK mRNA. PCK‐mRNA levels were monitored by Northern blot analysis and protein binding was analyzed by an electrophoretic mobility‐shift assay. In 24‐h cultured rat hepatocytes, binding of cytosolic protein to the PCK‐mRNA 3′ end and PCK‐mRNA levels were increased to a transient maximum at 2 h and 2–4 h, respectively, by a 1‐nM glucagon treatment, added with a change of medium. 100 nM insulin, added simultaneously with glucagon, reduced the glucagon‐induced maximum of protein binding by 80% and the increase of PCK mRNA by about 30%. In controls without hormonal treatment protein binding at 1 h was also increased; this increase was prevented by insulin. 100 nM insulin, added 1 h after glucagon, reversed protein binding to the 3′ end of PCK mRNA to nearly initial levels within 1 h and impaired the glucagon‐induced increase in PCK‐mRNA levels by 30%. The transcriptional inhibitor cordycepin, added 1 h after glucagon, did not prevent the further increase in glucagon‐enhanced protein binding nor its reversal by insulin. It did, however, prevent a further significant increase in PCK mRNA. Hormonally regulated protein binding could be localized to the 256 distal bases of the PCK‐mRNA 3′ end. The proximal 466 bases of the PCK‐mRNA 3′ end as well as the 1050 bases of the histone‐H1°‐mRNA 3′ end and the 1200 bases of the arylsulfatase‐A‐mRNA 3′ end also bound cytosolic protein(s), but this protein binding was not altered by treatment with glucagon or insulin. The 3′ end of PCK, arylsulfatase A and H1° mRNA exhibited strong binding of cytosolic protein(s) from diverse rat tissues such as heart, liver and lung as well as Fao rat hepatoma cells. Cytosolic protein(s) from spleen showed weak binding and proteins from HeLa and U937 tumor cells did not bind. Protein binding was most prominent with the 3′ end of PCK mRNA and cytosolic extracts from liver. The results support the hypothesis that the induction by glucagon and reversal by insulin of both protein binding to the 3′ end of PCK mRNA and the increase in PCK‐mRNA levels are correlated. This correlation suggests a functional relationship, possibly the regulation of mRNA stability by protein/mRNA interactions. Regulation of protein binding by glucagon and insulin is specific for the 3′ end of PCK mRNA and occurs possibly via covalent modification of the binding protein.