Regulation of tyrosine alpha-ketoglutarate transaminase in rat liver. 8. Inductions by hydrocortisone and insulin in cultured hepatoma cells.

Hydrocortisone addition to cell cultures of the Reuber (H-35) hepatoma initiated an 8to IO-fold increase in tyrosine transaminase (L-tyrosine:Z-oxoglutarate aminotransferase, EC 2.6.1.5) levels, the response being maximal at 9 hours and persisting at this new level for at least 36 hours. The optimal concentration of hydrocortisone for this induction was 5 X low7 M. Insulin was also found to elevate this enzyme in H-35 cells, and caused the enzyme to rise without the lag period that is apparent after hydrocortisone. With a single addition of insulin, the increase in enzyme (5to 6-fold) ceased after 8 hours, but a high steady state could be maintained by resupplementation with insulin. When the effect of insulin is measured at 6 hours, 1 milliunit per ml is optimal and 0.02 milliunit per ml is readily detected. The isolated A and B chains of insulin are not effective and do not compete with intact insulin. Glucagon is not effective in these cells, which appear to lack adenyl cyclase activity. Immunochemical-isotopic analyses show that both hydrocortisone and insulin specifically accelerate synthesis of the enzyme, but there may also be a stimulation of general protein synthesis after insulin treatment. Analysis of the kinetics of these inductions indicates a half-life of about 3 hours for tyrosine transaminase in the cells; this was contirmed by “chase” analysis and shown to be unaffected by insulin. Transaminases from hydrocortisone-induced, insulin-induced, and noninduced cells are immunologically identical with the rat liver enzyme.