Glucose-glucose 6-phosphate cycling in hepatocytes determined by incorporation of 3HOH and D2O. Effect of glycosyns and fructose.

The phosphorylation of glucose and recycling between glucose and glucose-6-P was determined in hepatocytes from fasted rats by a novel method. The cells were incubated with [U-14C]glucose as sole substrate in media containing 3HOH and D2O. Recycling was calculated from the yield of protons in glucose and glycogen. Results with 3HOH and D2O were identical. Phosphorylation was obtained as the sum of recycling plus the U-14C yields in products. At 10 mM glucose, more than 4 out of 5 molecules of glucose-6-P were recycled. About 1.2 mumol of glucose min/g of liver was phosphorylated, 1 mumol was recycled, and 0.2 mumol was glycolyzed. The effect of two phenacylimidazolium compounds (designated as glycosyns) and low concentrations of fructose (0.05-0.2 mM) on phosphorylation and recycling were examined. The glycosyns doubled glucose uptake, mainly as glycogen, nearly abolished glycolysis, and decreased recycling from 80 to 50-60%. There was little change in phosphorylation. Fructose doubled the yield of tritium from [2-3H]glucose in short term incubations (20-30 min), confirming the results of Van Schaftingen ((1993) Diabetologia 36, 582-588). The effect was transient, and cells became refractory to fructose. There was no glycogen synthesis and little effect on recycling. A new phenacylimidazolium compound stimulated glycogen synthesis and suppressed glycolysis and recycling, like the compound designated as proglycosyn by Yamanuchi et al. ((1992) Arch. Biochem. Biophys. 294, 609-615). This new compound (glycosyn-2) was fully active at lower concentrations (maximal effect at 0.02 mM).