Regulation of purine and pyrimidine metabolism by insulin and by resistance to tiazofurin

Abstract The purpose of this investigation was to clucidate the factors that regulate the pattern of gene expression in purine and pyrimidine metabolism in normal liver and hepatoma. For this purpose, the action of a hormone, insulin, and the development of resistance to a chemotherapeutic agent, tiazofurin, were studied. 1. 1.|This investigation brought detailed evidence showing that in the rat insulin exerted a profound effect on liver purine and pyrimidine metabolism by regulating the concentrations of nucleotides through controlling the activities of strategic enzymes involved in their biosynthesis. 2. 2.|When rats were made diabetic by alloxan treatment, in the average liver cell concentrations at ATP, GTP, UTP and CTP decreased to 66, 62, 54 and 63%, respectively, of those of normal liver. Administration of insulin for 2 days returned the hepatic nucleotide concentrations to normal range; further insulin treatment for an additional 5 days raised the concentrations of ATP, GTP, UTP and CTP to 197.352, 412 and 792% of values observed in the liver of diabetic rats. 3. 3.|In diabetic rats the hepatic activities of OMP decarboxylase, orotate phosphoribosyltransferase, uridine phosphorylase, uridine-cytidine kinase and uracil phosphoribosyltransferase decreased to 44, 48, 70, 36 and 41% of the activities of normalliver. Insulin treatment for 2 days returned activities to normal range. Continued insulin treatment for an additional 5 days increased the enzymic activities to 3.9- to 5.3-fold of those of the liver of the diabetic rats. The regulation by insulin treatment of the activities of enzymes of de novo and salvage synthesis of UMP should explain, in part at least, the decline and increase of the uridylate pool in diabetes and after insulin treatment. 4. 4.|In the diabetic rat hepatic CTP synthetase, the rate-limiting enzyme of CTP biosynthesis, decreased to 53% and insulin administration for 2 days restored activity to normal range. Insulin treatment for an additional 5 days increased the synthetase activity to 4-fold of the values of the diabetic liver. Thus, the behavior of liver CTP synthetase activity is tightly linked with that of the CTP pool. 5. 5.|In the diabetic rat liver, the activity of IMP dehydrogenase, the rate-limiting limiting enzyme of GTP biosynthesis, decreased to 24% of that of the normal liver. Insulin administration for 2 days returned the activity to normal range, yielding a 4.5-fold increase in the activity from the diabetic to the insulin-treated state. The decline of IMP dehydrogenase activity in diabetes and its restoration by insulin treatment indicates that the behavior of this enzyme is closely linked with that of the GTP pool. 6. 6.|Because the major action of insulin on liver has been thought to occur in carbohydrate metabolism, the impact of insulin administration in the rat was compared on carbohydrate-metabolizing and on purine and pyrimidine synthesizing liver enzymic activities. In the diabetic rat, the hepatic activities of glucokinase, pyruvate kinase and glucose-6-phosphate dehydrogenase decreased to 20, 30 and 65% of those of the normal rat liver. The hepatic enzymes of purine and pyrimidine biosynthesis declined to 54 to 85% with the exception of IMP dehydrogenase, carbamoyl-phosphate synthetase II and aspartate carbamoyltransferase which decreased to 14 to 28% of the activity of normal rat liver. Insulin administration restored enzymic activities to normal range in 2 days. The insulin-induced increases in the carbohydrate enzymic activities were 2.5- to 6.5-fold. A similar extent of increases was observed for most of the nucleic acid synthetic enzymes. These results indicated that some of the insulin-induced rises inhepatic enzymic activities in purine and pyrimidine biosynthesis are comparable to those for the classically recognized inductions in glycolytic and pentose phosphate enzyme activities. 7. 7.|The reprogramming of gene expression in cancer cells was examined in a variant of hepatoma 3924A cells in tissue culture which was resistant to the C-nucleoside, tiazofurin. In the hepatoma cells a biochemical program emerged that was characterized by a decrease in the transport of tiazofurin, in the content of TAD, the active metabolite of tiazofurin, and in the de novo synthesis of IMP to 2-, 2- and 4.7-fold of the activities of sensitive cells. By contrast, the pools of guanylates, the activities of IMP dehydrogenase, the guanylate salvageand that of the ratio of salvage to de novo synthesis were elevated 3-, 2.6-, 2.8- and 4.8-fold of those of the sensitive cells. Thus, the resistance to tiazofurin in hepatoma cells is expressed by an integrated reprogramming of gene expression.