Hormonal effects on glycogen metabolism in isolated hepatocytes of a freeze-tolerant frog

To determine whether specific hormonal responses were involved in the production of cryoprotectant (glucose) by liver of the freeze tolerant wood frog, Rana sylvatica, metabolically active hepatocytes were isolated in reasonable yields (mean 20.1 ± 1.30% SEM, n = 29) by in situ liver perfusion with collagenase. Freshly isolated cells from autumn-collected frogs contained large amounts of glycogen (650 μmol glucosyl units/g packed cells) and produced glucose from this endogenous reserve at a rate of 10 μmol g-1 hr-1 at 0°. Glucose output from cells was highly responsive to the addition of hormones; rates of glucose release increased 2.1-, 1.7-, and 1.7-fold with the addition of 10-7 M bovine glucagon, 10-7 M epinephrine, and 5 × 10-6 M dibutyryl-cyclic AMP, respectively. Norepinephrine, 5-hydroxytryptamine, and bovine insulin were without effect at 0.1 μM/l. Hormone stimulation of glucose release was correlated with an increase in both the total activity and the percentage a of glycogen phosphorylase in hepatocytes. However, none of the hormones tested affected the kinetic properties of hepatocyte pyruvate kinase, suggesting the absence of covalent modification control of the enzyme. The data indicate that the freezing-stimulated production of large quantities of glucose as a cryoprotectant by R. sylvatica liver does not involve qualitative differences in the hormonal control of liver glycogenolysis, compared with other lower vertebrates. However, quantitative differences were seen, such as the much greater phosphorylase activity, 4.38 ± 0.33 μmol min-1 g-1 packed cells, in freshly isolated R. sylvatica hepatocytes compared with 0.36 ± 0.06 μmol min-1 g-1 in Rana pipiens hepatocytes