An estimation of pyruvate recycling during gluconeogenesis in the perfused rat liver

Abstract

To estimate the degree of recycling of pyruvate during gluconeogenesis, an isotope tracer procedure was employed. Using the isolated, perfused rat liver with pyruvate-2- 14C in the perfusion fluid, the 3-carbon acids lactate and pyruvate were isolated and the distribution of 14C in each carbon was assayed. It can be shown that the degree of recycling can be approximated as twice the sum of 14C in carbons 1 and 3. Glucose, acetoacetate, and β-hydroxybutyrate were also determined, and their 14C distribution estimated by appropriate degradation procedures. In livers from fasted rats, recycling of pyruvate during 1 hr incubation occurred at a rate of 0.21 μmoles ± 0.02 (SE)/min/g while gluconeogenesis occurred at a rate of 0.49 ± 0.11 μmoles pyruvate-2- 14C/min/g. In livers from carbohydrate-fed rats, the ratio was reversed, with 0.35 ± 0.06 μmoles pyruvate-2- 14C recycled and only 0.09 ± 0.03 μmoles converted to glucose. These patterns were not affected by the simultaneous presence of octanoate in the perfusion, during which ketone body production was greatly increased. Only about 20% of the ketone bodies formed were derived from pyruvate, much less with octanoate present, and over 95% of the total radioactivity was in carbons 1 and 3 of acetoacetate as anticipated from the degree of pyruvate recycling. The glucose invariably had 3–4% of its total activity in carbons 3 and 4 and the remainder distributed approximately equally in carbons 1, 2, 5, and 6. The radioactivity in respired CO 2 indicated that about 13–25% of the total O 2 uptake was due to pyruvate oxidation to CO 2. The data indicate that during the course of liver perfusion a part of the pyruvate substrate is recycled back to pyruvate after conversion to 4-carbon acids. A major portion is recycled directly via the dicarboxylic acid cycle while a minor portion is recycled after traversing the tricarboxylic acid cycle. The extent of recycling was increased greatly by carbohydrate feeding while conversion to glucose was greatly reduced. Recycling may occur via three mechanisms: reversal of pyruvate carboxylase, the malic enzyme, and pyruvate kinase, but it is assumed that, the major path is via pyruvate kinase; and the results point to the possibility that the activity of this enzyme may regulate the rate of glucose production.