Cornell University Ithaca, New York, United States
Objectives: Our previous research has demonstrated that glutathione peroxidase-1 (GPX1) overexpressing (OE) mice developed nonalcoholic fatty liver disease (NAFLD), and the development was associated with increases in both hepatic mRNA and protein levels of SELENOK and(or) endoplasmic reticulum (ER) stress related factors. This study was to reveal if the elevated expression of SELENOK was induced by the overproduction of GPX1 protein or activity in the liver of mice.
Methods: In Experiment 1, liver tissues from the OE and their wild-type (WT) mice (n = 4, males, 6 months old) were collected and assayed for expression of SELENOK and ER stress related proteins (GRP78, IRE1a, and XBP1). In Experiment 2, GPX1 knockout (KO) and their WT mice (n = 3-5, male, 6 months old) were injected intraperitoneally with N-acetylcysteine [NAC, 50 mg/kg body weight, in phosphate-buffered saline (PBS)], GPX mimic ebselen [50 mg/kg, in dimethyl sulfoxide (DMSO)], diquat (10 mg/kg, in PBS) or their respective vehicle controls. Liver tissues were collected and assayed for SELENOK after 12 h. In Experiment 3, human GPX1 cysteine (Cys) mutant was cloned into pRetroX-Tight-Pur (A gift from Dr. Alan Diamond, University of Illinois-Chicago) and transfected into HepG2 cells. The cells were collected after 48 h and assayed for protein and(or) activity levels of GPX1 and SELENOK.
Results: Compared with the WT mice, the OE mice had hepatic SELENOK protein level elevated by 2.3-fold (P < 0.05), and protein levels of GRP78, IRE1a, and XBP1 elevated by 1.5-3.2-fold (P < 0.05). Compared with the respective vehicle controls, the ebselen and NAC injections enhanced (P < 0.05) in both genotypes, but the diquat injections decreased (P < 0.05) in the WT mice, hepatic SELENOK protein levels, respectively. Compared with the control, the transfection of the GPX1 Cys mutant in HepG2 cells caused no change in SELENOK protein expression when the GPX1 protein was elevated by 4.3-fold (P < 0.05).
Conclusions: The up-regulated hepatic SELENOK protein in the OE mice was unlikely to be driven by the GPX1 protein directly.
Funding Sources: This research did not receive external funding.
