Regulation of glycogen phosphorylase in fat body of Cecropia silkmoth pupae

Glycogen phosphorylase of pupal fat body of the silkmoth, Hyalophora cecropia, and its activation by different stimuli have been studied. Spectrophotometric assay in the direction of glycogenolysis, used in most of the experiments, indicated higher amounts of phosphorylase a than assay by release of P_i from glucose-1-phosphate; both assays, however, estimated changes in proportion of phosphorylase a equally. The K_ms for P_i were estimated as 5 mM for phosphorylase a in the absence of AMP and 18 mM for phosphorylase b with 2 mM AMP. When diapausing pupae were held at 4°C, fat body phosphorylase was quickly activated by conversion to the a form up to about 50% of the total, and then declined again after 30 days, when glycerol had accumulated in the hemolymph. Cold activation in vivo was quickly reversed at 25°C. Removal of the brain did not prevent cold activation. After storage at 15°C, sensitivity to cold activation was diminished. Locusts and crickets also showed activation of phosphorylase after chilling. Exposure of fat body to air, transfer to Ringer solution, or physical agitation, caused activation of phosphorylase which is classed as "shock" activation. After about 1 h incubation in Ringer at 25°C, this effect reversed spontaneously. Activation also occurred in fat body in vitro after transfer to 0°C ("cold" activation), and was reversed at 25°C. The previously reported inhibition of activation by glycerol, however, could not be consistently reproduced. In fat body homogenates, phosphorylase b was converted to phosphorylase a by incubation with ATP and Mg²⁺, which indicates activity of phosphorylase kinase. In preparations treated with Sephadex G-25 and then incubated, the reverse conversion took place, which was inhibited by fluoride, and indicates activity of phosphorylase phosphatase. Cyclic AMP added to fat body in vitro, or theophylline either in vivo or in vitro, stimulated the activation of phosphorylase. In fat body in vitro, shock activation was paralleled by elevation of tissue cyclic AMP, whereas cold activation was not. Cyclic GMP did not stimulate activation, and showed no significant changes in tissue levels. It is concluded that the conversion of silkmoth pupal fat body phosphorylase b to phosphorylase a can be stimulated by a shock-initiated mechanism involving cyclic AMP and a distinct cold-initiated mechanism independent of cyclic AMP.