Enzymes related to the
oxidative stress response, such as carbonyl reductase 3 (CBR3), or glutathione-S-transferase were more highly expressed in C3H/He mice on normal diet but were markedly increased in DDC-fed C57BL/6 mice. These data indicated profound alterations in basic biological processes in these mouse strains under basal conditions that could lay the foundation for different responses under disease conditions. Interestingly, some of these alterations (e.g., NDPK) were not seen at the messenger RNA (mRNA) expression level but only became evident after using a proteomics approach. Furthermore, in addition to the striking alteration of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression, aggregation and nuclear translocation of this enzyme were observed. NDPH and GAPDH are of particular interest in the context of MDB pathogenesis. NDPK catalyzes the transfer of the PLX4032 terminal phosphate of nucleoside triphosphates to nucleoside diphosphates, thereby being important for the synthesis of GTP, CTP, and UTP. NDPK (also called Nm23) has been identified as a metastasis suppressor whose function is regulated by oxidation and reduction at its Cys109.7 GAPDH is best known as a glycolytic “housekeeping” cytoplasmic enzyme.
However, it appears to be involved in several additional GSK126 concentration processes such as DNA repair, transfer RNA (tRNA) export, membrane fusion, cytoskeletal dynamics, and cell death.8, 9 These different functions are, at least in part, regulated by oxidative stress, resulting in posttranslational modifications, oligomerization, aggregation, and nuclear translocation of GAPDH. To further understand the biological relevance of the strain-specific alterations of NDPK and GAPDH, a series of in vitro and ex vivo 上海皓元医药股份有限公司 experiments were performed by Snider et al. Using the reactive oxygen species (ROS)-sensitive fluorescent probe CM-H2DCFDA they demonstrated that DDC resulted in higher ROS levels in cultured C57BL/6 than in C3H/He hepatocytes. This increase in ROS was paralleled by an increase in CBR3 immunoreactivity, resembling the in vivo situation observed in livers of mice.
Furthermore, DDC treatment of hepatocytes isolated from C57BL/6 mice resulted in a dose-dependent reduction of cytoplasmic GAPDH and nuclear translocation which could be inhibited by pioglitazone. Using small interfering RNA (siRNA)-mediated knockdown, GAPDH was identified as an upstream regulator of NDPK and other enzymes involved in antioxidant responses. Consequently, knockdown of GAPDH or NDPK resulted in increased ROS formation in hepatocytes. Interestingly, knockdown of NDPK resulted also in a decrease of GAPDH, suggesting a coregulation of these two enzymes. The effects of GAPDH knockdown were not restricted to antioxidant enzymes but also affected metabolic functions, e.g., by down-regulation of fumarylacetoacetate hydrolase. The work of Snider et al.