Abstract
Our previous studies1, 2) have covered key glycolytic and gluconeogenic enzyme activities in biopsied liver tissues obtained from the patients with liver diseases and in experimentally injured rat livers. It has been reported then that increased activities of low-Km hexokinase and glucose 6-phosphate dehydrogenase, which are nonspecific to liver, and decreased activities of glucokinase, glucose 6-phosphatase and fructose 1,6-diphosphatase, which are specific to liver, were observed in those injured livers. Thus, the altered enzyme pattern in liver injuries resembled that in undifferentiated cells, such as hepatomas, regenerating liver and fetal liver.
Similar studies were made of human and rat (male Sprague-Dawley) liver pyruvate kinase (PK), which exists in two isozyme forms, PK-L and PK-M2. Relative activities of these two isozymes in the rat were determined either by differential inhibition by 2.5mM ATP in the presence of 40mM MgCl2 (kinetic analysis), PK-L being inhibited and PK-M2 uninhibited, or by cellogel electrophoresis and densitometry. The latter method was used exclusively for determination of human liver PK isozyme activities. PK-L and PK-M2 activities were calculated on the basis of the relative activity ratios and the total PK activities.
PK-L, which is the liver-specific major component in fed rat and is considered to be a differentiated type in liver, decreased in activity by fasting and increased by refeeding glucose-casein, whereas PK-M2, which is the minor component and is considered as the prototype in liver, did not change in activity by dietary alteration but increased in fetal and partially hepatectomized rat livers, and AH 130 hepatoma. This confirms the results reported by other workers5, 6).
In several experimental liver injuries of rats, such as acute intoxication with CCl4 or thioacetamide, chronic intoxication with CCl4 and feeding on a choline deficient diet, the activity of PK-L was reduced, while that of PK-M2 significantly increased to result in an isozyme pattern similar to that observed with AH 1301). Following intraperitoneal administration of a single dose of 0.2ml of CCl4 per 100g body weight to the rat, the total PK and PK-M2 activities increased 4 and 10 fold, respectively, over the controls in 3 days and returned to the initial values in 1 week. In contrast, PK-L activity decreased by one third of the control value in 2 days and returned to the original level in 1 week. These alterations were more marked when the activities determined by the electrophoretic method were compared, because contamination of PK-L or PK-M2 with each other inherent to kinetic analysis could be avoided by this method.
Administrations of cycloheximide (100μg/100g body weight in 24, 27 and 30 hours after CCl4 treatment) suppressed the increase of PK-M2 activity in CCl4 liver injury, suggesting the requirement of de novo synthesis of PK-M2 protein for the increased activity. Involvement of humoral factor in the CCl2-mediated induction of PK-M2 was neglected from the results of parabiosis experiments.
PK-L and PK-M2 activities in biopsied human liver were studied in patients with various liver diseases. Although the total PK activity failed to show any significant differences among the patients with liver diseases studied, the activity of PK-L decreased and that of PK-M2 increased inversely in chronic hepatitis (active form), especially in that accompanied by sublobular hepatic necrosis and cirrhosis of the liver. The PK-L activities in fatty livers were significantly high. The altered pattern of PK isozymes in chronic hepatitis and cirrhosis of the liver resembled that in primary hepatoma.
