Regulation of glycogen synthase and glycogen phosphorylase by insulin in HepG2 cells
Syed, Noor Afshan
MetadataShow full item record
The effect of insulin on glycogen synthesis and glycogen metabolizing enzymes, glycogen synthase and glycogen phosphorylase, was studied in HepG2 cells. Glycogen synthesis was stimulated in HepG2 cells by 186%. Insulin (100 nM) caused a maximum 110% increase in glycogen synthase activity in 5 min. and a decrease of 65% in glycogen phosphorylase "'a'". There was a significant inverse correlation between activities of glycogen synthase "'a'" and glycogen phosphorylase "' a'" (R2 = 0.66, P < 0.001). Addition of 30 mM glucose caused decrease in phosphorylase "'a'" activity in the absence of insulin and this effect was additive with insulin up to 10 nM concentration. The activation of glycogen synthase by insulin was prevented by wortmannin but not by PD98059 or rapamycin. In fact, PD98059 slightly stimulated glycogen synthase activation by insulin. The inactivation of phosphorylase "'a'" by insulin was prevented by wortmannin and rapamycin but not by PD98059. Under these experimental conditions, insulin decreased glycogen synthase kinase-3β activity by 30-50% and activated more than 4-fold particulate protein phosphatase-1 activity and 1.9-fold protein kinase B activity; changes in all of these enzyme activities were abolished by wortmannin. The inactivation of GSK-3β and activation of PKB by insulin were associated with their phosphorylation and this was also reversed by wortmannin. The addition of protein phosphatase-1 inhibitors, okadaic acid and calyculin A, completely abolished the effects of insulin on both enzymes. These results indicate that in HepG2 cell, insulin regulates glycogen synthase and glycogen phosphorylase through (i) a common signalling pathway at least up to PI-3 kinase and bifurcates downstream; (ii) that PP-1 activity is essential for the effect of insulin and (iii) inactivation of glycogen phosphorylase by insulin is mediated through the PI-3 kinase pathway involving a rapamycin-sensitive p70s6k and PP-1G. Furthermore, this study also shows that in HepG2 cells, serine 473 phosphorylated PKBα is present in the nucleus of unstimulated cells and insulin stimulation results in translocation to the nuclear membrane. In contrast to this, in normal hepatocytes, phosphorylated PKBα is not found in unstimulated cells and upon stimulation with insulin it is translocated to the plasma membrane.