Regulation of glycogen phosphorylase in hypoxic cancer cells

Abstract

Compared to normal cells, many tumor cells have to subsist in a hypoxic intratumoral environment that has an unstable supply of oxygen and nutrients including glucose. How tumor cells may survive the metabolic stress arising from tumor hypoxia is not yet fully understood. Recent studies revealed that tumor cells are able to accumulate large quantities of intracellular glycogen. Whether glycogen would serve as fuel reserve in hypoxic tumor cells is presently not clear. This question is being addressed in this study.

When HeLa, HT29, HEK293 and HepG2 cells were incubated under hypoxic condition in the absence of glucose, the steady state intracellular glycogen level dropped by more than 50% in 3 hours. The specific pharmacological inhibition of the liver isoform glycogen phosphorylase (PYGL) (CAS 648926-15-2) partially inhibited hypoxia-induced glycogen degradation. More complete inhibition was achieved by combined incubation using the pharmacological inhibitor and 2-deoxyglucose. Inhibition of glycogen degradation resulted in decrease in hypoxia-induced lactate formation, supporting the idea that glycogen serves as a fuel reserve in hypoxic cancer cells.

Inhibition of autophagy or alpha-glucosidase failed to prevent glycogen degradation in hypoxic condition, suggesting that cytosolic glycogen phosphorylase is the major enzyme involved in glycogen degradation. The mRNA, protein and phosphorylation levels of glycogen phosphorylase were unaltered by hypoxia. The siRNA-mediated knockdown of the brain form of glycogen phosphorylase (PYGB) resulted in markedly greater inhibition of glycogen degradation than did the knockdown of PYGL. Whereas the enzyme activity of PYGB can be markedly stimulated by AMP, the activity of PYGL is only slightly stimulated in the presence of AMP. The relative proportion of AMP-sensitive and AMP-insensitive GP activity is little affected by acute hypoxia.

In conclusion, direct evidence is provided in this study that glycogen may serve as an intracellular fuel reserve in tumor cells. The involvement of the brain form of glycogen phosphorylase is for the first time demonstrated to be involved in the mobilization of this fuel reserve in tumor cells.