DEMONSTRATION OF A GLYCOGEN/GLUCOSE 1-PHOSPHATE CYCLE IN HEPATOCYTES FROM FASTED RATS - SELECTIVE INACTIVATION OF PHOSPHORYLASE BY 2-DEOXY-2-FLUORO-ALPHA-D-GLUCOPYRANOSYL FLUORIDE

In search for a nonmetabolized, superior glucose analogue to study the mechanism of glucose-induced glycogen synthesis, we have tested 2-deoxy-2-fluoro-alpha-D-glucopyranosyl fluoride, which inhibits muscle phosphorylase b 10 fold better than does glucose (Street, I. P., Armstrong, C. R., and Withers, S. G. (1986) Biochemistry 25, 6021-6027), In a gel-filtered liver extract, 0.6 mM analogue and 10 mM glucose equally accelerated the inactivation of phosphorylase and shortened the latency before the activation of glycogen synthase, The analogue was not measurably defluorinated or phosphorylated by intact hepatocytes, as monitored by F-19 NMR. When added to isolated hepatocytes, 10 mM analogue inactivated phosphorylase more extensively than did 50 mM glucose, but unlike glucose, it did not result in the activation of glycogen synthase, Therefore, the binding of glucose to phosphorylase a can account for the inactivation of phosphorylase, but the metabolism of glucose (probably to Glc-6-P) appears to be required to achieve activation of glycogen synthase. The livers of overnight-fasted, anesthetized mice contained appreciable amounts of both phosphorylase a and glycogen synthase a, without net glycogen accumulation, Likewise, hepatocytes isolated from fasted rats and incubated with 10 mM glucose contained 41% of phosphorylase and 32% of glycogen synthase in the a form, and these values remained stable for 1 h, while glycogen accumulated at only 22% of the rate expected from the glycogen synthase activity, The addition of 10 mM analogue decreased phosphorylase a to 10% without significant change in glycogen synthase a (38%), but with a 4-fold increased rate of glycogen accumulation. These findings imply that synthase a is fully active in the liver of the fasted animal and that the absence of net glycogen synthesis is due to continuous glycogenolysis by phosphorylase a.