Inhibition of hepatic gluconeogenesis by the Rp-diastereomer of adenosine cyclic 3′,5′-phosphorothioate

Abstract

The specific intracellular cyclic AMP-dependent protein kinase antagonist, the Rp-diastereomer of adenosine cyclic 3′,5′-phosphorothioate (Rp-cAMPS), inhibited both basal and cyclic AMP-agonist-induced rates of gluconeogenesis in hepatocytes isolated from fasted rats. Incubation of the cells in the presence of pyruvate and lactate and either the Sp-diastereomer of adenosine cyclic 3′,5′-phosphorothioate (Sp-cAMPS) or glucagon produced a concentration-dependent increase in the rate of gluconeogenic glucose production which was shifted to higher concentrations of Sp-cAMPS or glucagon in the presence of Rp-cAMPS. Incubation of the cells with Rp-cAMPS in the absence of agonist produced no increase in the rate of glucose production and, in most cases, 100 microM-Rp-cAMPS resulted in 14-20% decrease in the substrate-stimulated rate of glucose production. Sp-cAMPS-induced gluconeogenesis was inhibited half-maximally at 1 microM-Rp-cAMPS and glucagon-induced gluconeogenesis was inhibited half-maximally at 12 microM-Rp-cAMPS. Approx. 10-15% of the inhibition of gluconeogenesis observed in the presence of Rp-cAMPS was due to conversion of glucose 6-phosphate to liver glycogen, consistent with Rp-cAMPS-induced reactivation of glycogen synthase. The remaining 85-90% inhibition of gluconeogenic glucose production resulted from the action of Rp-cAMPS on the cyclic AMP-sensitive enzymes controlling the rate of gluconeogenesis.

A mechanistic and kinetic analysis of the interactions of the diastereoisomers of adenosine 3′,5′-(cyclic)phosphorothioate with purified cyclic AMP-dependent protein kinase

“…Although they differ structurally from one another only in the orientation of the sulphur atom with respect to the chiral phosphorus atom, cAMPdependent protein kinase, the target enzyme, recognizes them as different molecules. Thus the analogues have a 10-fold difference in binding affinity for the holoenzyme as measured by [3H]cAMP displacement and opposite effects on enzyme activity measured in vitro as phosphotransferase activity (De Wit et al, 1982, 1984 and in isolated hepatocytes as glucose production (Rothermel et al, 1983(Rothermel et al, , 1984aDragland-Meserve et al, 1986;Marks & Parker Botelho, 1986). Sp-cAMP [S], which has an axial sulphur atom, binds to the holoenzyme with a 10-fold lower affinity compared with cAMP and promotes dissociation of the holoenzyme.…”