Objective: The purpose of this study is to observe whether the glycolysis bypass is activated under high-Se stress.
Method: Firstly, five different concentrations of selenomethionine (SeMet, 0.001, 0.01, 0.1, 1 and 10 mmol/L) were added into the three kinds of cell lines (L02, HepG2 and HCT-116) and incubated for 48 hours. The expressions of selenoproteins (GPX1 and SELENOP) and key enzymes for the de novo serine biosynthesis (PHGDH) or synthetic serine as the one carbon unit donor (SHMT1) were analyzed by ELISA and WB to screen the most sensitive cell line. Then, with more detailed levels of SeMet (0.001, 0.005, 0.01, 0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 1, and 10 µmol/L) given, L02 cell was cultured for 48 hours to analyze the trends of selenoproteins and serine-related metabolic enzymes by WB. Finally, the impact of exogenous serine or glycine on the expressions of selenoproteins and metabolic enzymes were evaluated in L02 cell cultured in high-Se media.
Result: Unlike in HepG2 cell and HCT-116 cell, the expression trend of selenoproteins and key metabolic enzymes with increased SeMet concentration is similar in L02 cells, which is a parabola with slight differences at the top. The corresponding SeMet concentration for the inflection point of GPx1, SELENOP, PHGDH and SHMT1 is 0.075µmol/L, 0.05µmol/L, 0.05µmol/L and 0.05µmol/L respectively. Also in L02 cell cultured in high-Se media (added with 0.05µmol/L SeMet), there were dose-dependent feedback inhibition effects of exogenous serine or glycine on the expressions of PHGDH. Meanwhile there were promotional effects of exogenous serine or glycine on the expressions of GPx1 and SELENOP.
Conclusions: The L02 cell line is the most suitable for the studies of selenoprotein expression and regulation in vitro among the three kinds of cell lines. Under high-Se stress, the serine de novo synthetic pathway might be utilized to synthesize more endogenous serine and could be feedback inhibited by exogenous serine or glycine. Further research works need to be taken based on the well-tested high-Se induced IR animal models.