Effect of human serum on the expression levels of the d-aminolevulinate synthetase 1 protein in C3A hepatoma cells

Abstract

Studies carried out in our laboratory, in an in vitro model, demonstrated that the presence of fetal bovine serum (FBS) in the cultura medium caused an increase in the amount of δ-aminolevulinate synthetase (ALA-S1) protein, the first and regulatory enzyme of the heme pathway, due to an increase in protein translation. It has also been described that the addition of insulin influences markedly the induction of ALA-S1 through the PI3K / Akt pathway. Therefore, in order to continue studying in vitro the effects obtained with FBS and to extend the research to human serum (Sh), it was decided to evaluate the effects of serum in cultures of the established line of human hepatoma C3A.The cells were grown in low glucose DMEM medium (5 mM) and without serum for 18 h. The cells were then treated with different concentrations of Sh (1, 2.5, and 10%), with 2% Sh inactivated (Shi) by heat or stripped with activated carbón (SHe) and 2% FBS. The addition of Sh caused a significant increase in ALA-S1 protein (100%) at all concentrations. Heat inactivation or activated carbon treatment had no effect on the induction of ALAS1 by Sh. However, the expression levels of ALA-S1 mRNA were significantly decreased (30%). This result agrees with the observed increase of the phosphorylation levels of Akt (Ser473), an enzyme that inactivates the transcription factor needed for the transcription of ALA-S1 mRNA. The phosphorylation levels of 4-EBP1 were 172% increased. When 4-EBP1 is phosphorylated, the protein synthesis is favored, thus the obtained result would suggest that the increase in ALA-S1 protein is due to a greater translation. These results here presented constitute a significant contribution to the understanding of the molecular mechanisms that lead to changes in the expression of ALA-S1. Moreover, the in vitro model used is adequate to further research the effect of Sh on ALA-S1.