The activation of glycogen synthase in hepatocytes from rats with a glycogen storage disorder (gsd/gsd).

Rats from the NZR/Mh strain have a genetically-determined deficiency of phosphorylase b kinase activity in the liver which in homozygotes &d/g@ produces a poor activation of glycogen phosphorylase and very high liver glycogen concentrations [I]. They also have low active glycogen synthase activities [l] and previous studies on liver homogenates have shown an inhibition of glycogen synthasephosphatase by high glycogen concentrations [2]. This appears to be the major control of glycogen synthesis in liver of gsd/g.sd and fed normal animals [2] rather than the control of glycogen synthasephosphatase by active glycogen phosphorylase [3]. It is of interest to know if, and to what extent, glycogen synthase in gsd/gsd liver can be activated. Here, we describe the effects of glucose on the active forms of glycogen synthase and phosphorylase in hepatocytes from gsd/gsd and normal livers. In gsdlgsd cells with high glycogen levels, the activation of glycogen synthase in response to 80 mM glucose was blunted despite the very low active phosphorylase activities and was inversely correlated with the glycogen content of the cells. The findings do not support the concept of phosphorylase inactivation being a prerequisite for activation of glycogen synthase [3]. 7.3 mM glucose, 3.3 mM pyruvate, 2.3 mM glutamate with 0.3 mg/ml collagenase (Sigma Chemical Co., St Louis MO). After 30 min, the liver was minced and incubated for 15 min at 37°C in a medium containing 2.5 mM calcium, 25 mM glucose and 1.5% gelatin in the above buffer. The dispersed cells were then filtered through gauze and washed 3 times with glucose-free incubation medium. Cell suspensions were diluted 1: 10 with incubation medium and preincubated at 37°C for 15 min with constant oxygenation (100% 02) before incubation with glucose. Incubations were started by adding cell suspension to flasks containing glucose at lo-80 mM final cont. Following incubation, 0.5 ml aliquots for assay of enzymes and glycogen were added to 0.5 ml 200 mM Tris-HCl (pH 7.8) containing 20 mM EDTA, 200 mM NaF and 20 mM cysteine and frozen in an alcohol/solid CO2 bath. Aliquots from each flask were also spotted on millipore filter discs under suction and dried to constant weight for determination of a dry cell weight.