Hormonal Regulation of Rat Liver Cells Cultured in Chemically Defined Medium

Abstract

The specific activity of the enzyme tyrosine 2‐oxoglutarate transaminase from an established rat liver‐cell line grown in chemically defined medium, increases under the addition of the synthetic glucocorticoid dexamethasone sodium phosphate. Insulin also induces an enzyme increase but only in cells exposed previously to dexamethasone. Lower and ineffective glucocorticoid concentrations are, however, effective per se in making the cells insulin‐sensitive. Optimal concentrations of the glucocorticoid increase the enzyme activity 3 to 4‐fold during the first 8–9 h of incubation, however the cells are not insulin‐sensitive until the tenth hour of incubation. These findings suggest that dexamethasone induces a factor(s) necessary for the insulin effects only on the enzyme since insulin increased the rate of labeled amino‐acids incorporation in trichloroacetic acid precipitates without need for cell pretreatment with the other hormone. Dexamethasone was found to increase the rate of labeled uridine incorporation into trichloroacetic acid preeipitates. Experiments using RNA and protein‐synthesis inhibitors indicate that dexamethasone operates at a transcriptional level, while insulin works at a translational level. Actinomycin does not “superinduce” the enzyme, however when the cells are preincubated with medium plus serum the above mentioned phenomenon reappears. At least two forms (I and II) of the enzyme have been observed in the cells by hydroxyapatite column chromatography using a phosphate step gradient. Both forms are inducible by dexamethasone. Form I is extremely heat‐sensitive (65 °C) while form II is completely heat‐resistant in the same condition. Incubation of the cells with dexamethasone does not change the heat stability of form II but increases significantly the resistance of form I to heat treatment. Insulin appears not to affect the heat stability of the enzyme.