The Effects of Glucose Concentration Change during Cell Culture on O-GlcNAcylation and Phosphorylation of Akt

Abstract

O-GlcNAc modification is a glycosylation occuring on proteins in nucleus and cytosol. It is one of the post-translational modifications that regulates many cellular processes including the expression and the function of various proteins. The sugar donor of O-GlcNAc, UDP-GlcNAc, is synthesized from glucose through HBP(Hexosamine Biosynthetic Pathway) and its synthesis is highly influenced by the extracellular glucose concentration. Considering that O-GlcNAc level changes according to the glucose concentration, we focused on the change of glucose concentration in the media during culture duration. Here, we investigated what effects this change has on many cellular processes. First, we measured how the glucose concentration in the media changes during culture duration. Cells were cultured in both euglycemic(5mM) and hyperglycemic(25mM) media to find out not only the change of the concentration, but also the effects of starting concentration in cell culture system. As time passed, the glucose concentration in the media decreased in both conditions. Especially, in the case of cancer cells which have highly active metabolism, the final concentration was even lower than the half of the starting concentration. With this result, O-GlcNAc level also generally decreased and was higher in high-glucose condition than normal-glucose condition. To find out the effects of these different O-GlcNAc levels, a comparison was made between glucose-concentration-maintained sample and non-maintained sample. As a result, Akt, an important kinase involved in insulin signal transduction, was found to be less phosphorylated(Ser473) in glucose-maintained condition than non-maintained condition. Akt was also found to be more O-GlcNAcylated in glucose-maintained condition. Moreover, the phosphorylation of GSK-3β, the downstream protein of Akt, reduced in glucose-maintained condition. These results suggest that O-GlcNAcylation becomes different by the initial concentration and the decrease of glucose in the media during culture duration. In addition, the change in O-GlcNAc affects the signaling pathways in the cell. The glucose condition and its maintaining should be considered in the culture system, especially in the field of studying glycosylation and phosphorylation.