EFFECTS OF C-1-SUBSTITUTED GLUCOSE ANALOG ON THE ACTIVATION STATES OFGLYCOGEN-SYNTHASE AND GLYCOGEN-PHOSPHORYLASE IN RAT HEPATOCYTES

A series of glucose-analogue inhibitors of glycogen phosphorylase b (G Pb) has been designed, synthesized and investigated in crystallographi c binding and kinetic studies. The aim is to produce epsilon compound that may exert more effective control over glycogen metabolism than th e parent glucose molecule and which could alleviate hyperglycaemia in Type-II diabetes. N-Acetyl-beta-D-glucopyranosylamine (1-GlcNAc) has a K(b)oth enzymes were stronger than those of glucose, with 0.8 mM 1-Gl cNAc being equipotent with 50 mM glucose. At 1 mM, 1-GlcNAc enhanced t he dephosphorylation of exogenous GPa by liver extracts (600%) and by muscle extracts (75%). This represents an approximately 500-fold impro vement on glucosei for muscle GPb in elude extracts of 30 mu M, 367-fo ld lower than that of beta-D-glucose [Board, Hadwen and Johnson (1995) fur. J, Biochem. 228, 753-761]. In the current work, the effects of 1 -GlcNAc on the activation states of GP and glycogen synthase (GS) in c ell-free preparations and in isolated hepatocytes are reported, In gel -filtered er;tracts of liver, which lack ATP for kinase activity, 1-Gl cNAc produced a rapid and time-dependent inactivation of GP with;I sub sequent activation of GS. Effects of 1-GlcNAc on for the liver activit y and 40-fold for the muscle activity. In whole hepatocytes, 1-GlcNAc showed an approximately 5-fold enhancement of glucose effects for GP i nactivation but failed to elicit activation of GS. Glucose-induced act ivation of GS in whole hepatocytes was reversed by subsequent addition of 1-GlcNAc. However, when GS activation was achieved via the adenosi ne analogue and kinase inhibitor, 5'-iodotubercidin (ITU), subsequent addition of 1-GlcNAc allowed continued activation of GS. Phosphorylati on of 1-GlcNAc in rat hepatocytes was established using radiolabelled material. The rate of phosphorylation was 1.60 nmol/min per 10(6) cell s at 20 mM 1-GlcNAc but was reduced by the presence of 50 mu M ITU (0. 775 nmol/min per 10(6) cells). It is suggested that the phosphorylated derivative of 1-GlcNAc formed in hepatocytes is 1-GlcNAc 6-phosphate and that the presence of this species is responsible for the failure o f 1-GlcNAc to activate GS. The relative importance of the reduction in concentration of GPa versus increased glucose 6-phosphate levels for activation of GS is discussed.