Glutathione peroxidase 4 and vitamin E cooperatively prevent hepatocellular degeneration

Abstract

The selenoenzyme glutathione peroxidase 4 (Gpx4) is an essential mammalian glutathione peroxidase, which protects cells against detrimental lipid peroxidation and governs a novel form of regulated necrotic cell death, called ferroptosis. To study the relevance of Gpx4 and of another vitally important selenoprotein, cytosolic thioredoxin reductase (Txnrd1), for liver function, mice with conditional deletion of Gpx4 in hepatocytes were studied, along with those lacking Txnrd1 and selenocysteine (Sec) tRNA (Trsp) in hepatocytes. Unlike Txnrd1- and Trsp-deficient mice, Gpx4^-/- mice died shortly after birth and presented extensive hepatocyte degeneration. Similar to Txnrd1-deficient livers, Gpx4^-/- livers manifested upregulation of nuclear factor (erythroid-derived)-like 2 (Nrf2) response genes. Remarkably, Gpx4^-/- pups born from mothers fed a vitamin E-enriched diet survived, yet this protection was reversible as subsequent vitamin E deprivation caused death of Gpx4-deficient mice ~4 weeks thereafter. Abrogation of selenoprotein expression in Gpx4^-/- mice did not result in viable mice, indicating that the combined deficiency aggravated the loss of Gpx4 in liver. By contrast, combined Trsp/Txnrd1-deficient mice were born, but had significantly shorter lifespans than either single knockout, suggesting that Txnrd1 plays an important role in supporting liver function of mice lacking Trsp. In sum our study demonstrates that the ferroptosis regulator Gpx4 is critical for hepatocyte survival and proper liver function, and that vitamin E can compensate for its loss by protecting cells against deleterious lipid peroxidation.

Keywords: Ferroptosis; Lipid peroxidation; Selenoprotein.

Graphical abstract

Fig. 1

Liver necrosis in Alb-cre; Gpx4^fl/fl mice. H&E staining of Gpx4 control and Alb-cre; Gpx4^fl/fl livers at 10× and 40× magnification from 1 day old pups revealed extensive hepatocyte degeneration and necrosis, lack of veins (arrow) and sinusoids in livers of Gpx4-deficient pups.

Fig. 2

Expression levels of selenoproteins in livers of 1 day old control, Trsp-, Txnrd1- and Gpx4-deficient mice. (A) mRNA levels of Gpx1, Gpx2, Gpx4, Txnrd1, Selr, and Sepw were analyzed by qPCR. Data are shown as relative mRNA levels normalized to Gapdh in all liver samples (n=3 for each genotype). *Denotes statistical difference (P<0.05). (B) Protein levels of Gpx1, Gpx4, Txnrd1, SelR and SepW were analyzed by immunoblotting in both control and knockout liver samples (n=2 each). Gapdh levels are used as a control for protein loading. (C) Txnrd1 activity in livers of control, Trsp-, Txnrd1- and Gpx4-deficient mice. Data are the mean±SEM for three independent experiments and are expressed as µmol TNB/min/mg protein. *Denotes statistical difference (P<0.05).

Fig. 3

mRNA expression levels of Nrf2-regulated genes in livers of 1 day old control, Trsp-, Txnrd1- and Gpx4-deficient mice. (A) mRNA levels of Gsta1, Gclc, Gsr, Srxn1 and Cbr3 were analyzed by qPCR. Data are shown as relative mRNA levels normalized to Gapdh in all liver samples (n=3 for each genotype). *Denotes statistical difference (P<0.05). (B) Total GSH was measured spectrophotometrically and the levels calculated relative to the levels found in control mice. Total GSH concentrations are the mean±S. D. for three independent experiments.

Fig. 4

Survival rate of Gpx4 control and Alb-cre; Gpx4^fl/fl mice following removal of vitamin E from the diet. (A) Schematic representation of experimental design and feeding plan of mice maintained on vitamin E-deficient (−vitamin E) or vitamin E-supplemented (+vitamin E) diets. (B) Survival curve of control (n=12) and Alb-cre; Gpx4^fl/fl (n=10) mice on defined diets. Arrow notes the time when mice were placed on a vitamin E-deficient diet. A log-rank test was used for statistical analysis. *Denotes statistical difference (P<0.0001).

Fig. 5

Liver necrosis in Alb-cre; Gpx4^fl/fl mice following removal of vitamin E. (A) H&E staining of Gpx4 control and two Alb-cre; Gpx4^fl/fl livers at 10× and 20× magnification from mice that have been maintained on a vitamin E-sufficient diet for 6 weeks, followed by removal of vitamin E for 3 weeks. N=areas of necrosis. Arrows point to areas of acute inflammation. (B) TBARS assay was used to assess plasma levels of malondialdehyde (MDA) in Gpx4 control (n=3) and Alb-cre; Gpx4^fl/fl (n=3) mice maintained on a vitamin E-supplemented diet (vitamin E) for nine weeks and in Gpx4 control (n=3) and Alb-cre; Gpx4^fl/fl (n=3) mice maintained on a vitamin E-supplemented diet for six weeks followed by removal of vitamin E (- vitamin E) for 3 weeks. *Denotes statistical difference (P<0.05).

Fig. 6

Survival curve of combined Trsp- and Txnrd1-deficient mice. Survival curve of control (n=10), Alb-cre: Trsp^fl/fl; Txnrd1^fl/fl(n=6), Alb-cre: Trsp^fl/fl; Txnrd1^fl/+ (n=6) and Alb-cre: Trsp^fl/fl; Txnrd1^+/+ mice (n=10) maintained on standard chow. A log-rank test was used for all statistical analyses. *Denotes statistical difference (P<0.0001) from control.