Regulation of Tyrosine Aminotransferase by Insulin in Cultured Rat Hepatoma Cells1

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Abstract
Insulin induced tyrosine aminotransferase (TAT) activity in cultured rat hepatoma cells (R117–21B). The induced enzyme activity reached a maximum at 6 to 8 hand then decreased to the control level after 24 to 48 h of incubation. The degree of the induction of TAT was related to the insulin concentration in the range of 0.005 to 0.5 ngJml, and above a concentration of 0.5 ng/ml the enzyme activity reached a plateau at 6 h of incubation. However, the binding of insulin to its receptors increased continuously after the maximum induction was observed. The number of high affinity insulin binding sites in R117–21B cells was 2.5 × 105 per cell, and the apparent dissociation constant (Kd) of the insulin receptor system at 25°C was 3.7 n It was revealed that, when only 1.9% of the total high affinity insulin binding sites were occupied by insulin in R117–21B cells, a maximum response in terms of enzyme induction occurred. Under these conditions, 3,900 insulin molecules were taken up by each cell to yield the maximum induction of TAT. When insulin alone was readded to the culture after disappearance of the insulin effect, the enzyme activity increased only slightly or did not change. However, if insulin was added in a fresh culture medium, the enzyme activity increased normally. This indicates that a certain component(s) in the conditioned medium inhibited the reinduction of TAT by fresh insulin. However, when insulin-untreated fresh cells were incubated with insulin in the conditioned medium, the induction of TAT was observed. These data show that some cellular modulation occurred during the insulin treatment as well, and that this modulation and the certain component(s) contained in the conditioned medium inhibited the reinduction of TAT activity by fresh insulin. The magnitude of the inhibitory effect of the conditioned medium on the reinduction of TAT by fresh insulin was related to the ratio of the conditioned medium to fresh medium during the induction of the enzyme. The unknown component(s) contained in the conditioned medium was heat-stable and dialyzable, and was not extracted with ethanol or acetic acid from the lyophilized conditioned medium. It was suggested, therefore, that insulin induces intracellular modulations in the hepatoma cells, and that the unknown inhibitory component(s) contained in the conditioned medium also contributes to the regulation of TAT by insulin.