Role of insulin, glucose, and cyclic GMP in the regulation of glucokinase in cultured hepatocytes.

Following insulin addition to the medium of primary cultures of hepatocytes, the activity of glucokinase increased by 50% during the first 4 h, remained constant for an additional 2 to 3 h, and was followed by a second increase in activity. This biphasic increase in activity was a reflection of the amount of glucokinase enzyme protein present as measured by quantitative immunoprecipitation. The level of cGMP was found to increase sharply 6 h following the addition of insulin and just prior to the second phase of the increase in glucokinase activity. When hepatocytes were cultured in the absence of carbohydrates, only the initial phase of the increase in enzyme activity in response to insulin was observed, and no increase in the level of cGMP was found. However, the addition of 8-bromo-cGMP to the culture medium at 6 h or later could restore the second increase in activity. The addition to the medium of glucose, fructose, pyruvate, or lactate enhanced the insulin-induced increase in glucokinase, but only fructose, pyruvate, and lactate increased the activity of the enzyme in the absence of insulin. When the phosphorylation of hexoses was blocked by the addition of N-acetylglucosamine to the culture medium, an intermediate level of glucokinase activity was observed in response to insulin. The full response was restored by the inclusion of 8-bromo-cGMP in the medium. Both phases of the increase in glucokinase activity were inhibited by cycloheximide, whereas the addition to the culture medium of cordycepin or actinomycin D prevented only the initial increase in activity and not the second increase, which arose from the addition of 8-bromo-cGMP to the cultures.