Regulation of selenoproteins and methionine sulfoxide reductases A and B1 by age, calorie restriction, and dietary selenium in mice

Abstract

Methionine residues are susceptible to oxidation, but this damage may be reversed by methionine sulfoxide reductases MsrA and MsrB. Mammals contain one MsrA and three MsrBs, including a selenoprotein MsrB1. Here, we show that MsrB1 is the major methionine sulfoxide reductase in liver of mice and it is among the proteins that are most easily regulated by dietary selenium. MsrB1, but not MsrA activities, were reduced with age, and the selenium regulation of MsrB1 was preserved in the aging liver, suggesting that MsrB1 could account for the impaired methionine sulfoxide reduction in aging animals. We also examined regulation of Msr and selenoprotein expression by a combination of dietary selenium and calorie restriction and found that, under calorie restriction conditions, selenium regulation was preserved. In addition, mice overexpressing a mutant form of selenocysteine tRNA reduced MsrB1 activity to the level observed in selenium deficiency, whereas MsrA activity was elevated in these animals. Finally, we show that selenium regulation in inbred mouse strains is preserved in an outbred aging model. Taken together, these findings better define dietary regulation of methionine sulfoxide reduction and selenoprotein expression in mice with regard to age, calorie restriction, dietary Se, and a combination of these factors.

FIG. 1.

Expression profiles of selenoproteins and MsrA in mouse organs and tissues. Equal amounts (10 μg of protein per lane) from indicated tissues of a 10-month-old male Balb/c mouse were loaded on SDS-PAGE gels, transferred onto PVDF membranes, and probed with anti-MsrB1, MsrA, GPx1, and TR3 antibodies.

FIG. 2.

Regulation of MsrB1 and MsrA expression and MsrB and MsrA activities in Balb/c mice by dietary selenium and aging. (A and B) Western blot analyses of liver samples from mice subjected to (-)Se, 0.1 ppm Se, and 0.4 ppm Se diets are shown for 10-month- (A) and 30-month-old (B) animals. The proteins probed are indicated on the left and protein loading is shown by Amido Black staining on bottom images. MsrB1 expression in female mice fed 0.1 ppm Se showed higher variability than in other samples. Also, in general, we observed variability from animal to animal, which is not related to activity and Western analyses. Specific MsrB (C) and MsrA (D) activities in the same set of samples (n = 36, two-way ANOVA interaction, strong evidence for the effect of diet on MsrB1 (p_MsrB1 < 0.007) and suggestive evidence for the effect on MsrA (p_MsrA < 0.06) activities). (E) Specific MsrB, MsrA, and total Msr activities in liver lysates in 5-month-old mice subjected to indicated selenium diets (n = 16, one-way ANOVA, p < 0.005). Total Msr activity represents a separate measurement with mixed methionine (R,S) sulfoxide substrate rather than the sum of MsrA and MsrB activities.

FIG. 3.

Regulation of MsrB1 and MsrA expression and MsrB and MsrA activities in Balb/c mice by a combination of dietary selenium and calorie restriction. (A) Western blot analyses in liver samples, with the proteins probed indicated on the left. These are calorie restriction (CR) samples (compare with Fig. 2A where regular food samples are probed). (B) Comparison of MsrB1, MsrA, GPx1, and TR3 expression in livers of 10-month-, 30 month-old, and CR mice. These mice were fed 0.4 ppm Se diet. The proteins probed are indicated on the left and protein loading is shown by Amido Black staining on bottom images of A and B. Specific activities of MsrB1 (C) and MsrA (D) in 10-month-old and CR animal groups. There was an effect of the diet on both MsrA and MsrB1 activity (n = 43, two-way ANOVA interaction, p_MsrA < 0.06 and p_MsrB1 < 0.05); however, the effect of CR was not significant (p_MsrA > 0.4 and p_MsrB1 > 0.12).

FIG. 4.

Regulation of MsrB1 and MsrA expression and MsrB and MsrA activities by dietary selenium in TGFα and TGFα/i6A^- transgenic mice. (A) Western blot analysis of proteins from TGFα (left panel) and TGFα/i6A^- (right panel) mice. The bottom image shows protein staining with Amido Black as a control of protein loading. Analysis of MsrB (B) and MsrA (C) activities in transgenic animals (n = 16, two-way ANOVA). There was an effect of selenium diet on MsrB activity, p < 0.04. However, no evidence was found against the null hypothesis for MsrA activity (p _MsrA > 0.28). There was also no significant difference in MsrA and MsrB activities in TGFα and TGFα/i6A^- transgenic mice (p_MsrA > 0.26, p_MsrB1 > 0.23).

FIG. 4.

Regulation of MsrB1 and MsrA expression and MsrB and MsrA activities by dietary selenium in TGFα and TGFα/i6A^- transgenic mice. (A) Western blot analysis of proteins from TGFα (left panel) and TGFα/i6A^- (right panel) mice. The bottom image shows protein staining with Amido Black as a control of protein loading. Analysis of MsrB (B) and MsrA (C) activities in transgenic animals (n = 16, two-way ANOVA). There was an effect of selenium diet on MsrB activity, p < 0.04. However, no evidence was found against the null hypothesis for MsrA activity (p _MsrA > 0.28). There was also no significant difference in MsrA and MsrB activities in TGFα and TGFα/i6A^- transgenic mice (p_MsrA > 0.26, p_MsrB1 > 0.23).

FIG. 5.

Regulation of MsrB1 and MsrA expression and MsrB and MsrA activities in HET outbred mice. (A) Female mice were subjected to six indicated diets (four animals per diet). Their liver samples were analyzed by Western blotting, as indicated. The proteins probed are indicated on the left and protein loading is shown by Amido Black staining on bottom image. Analysis of MsrB (B) and MsrA (C) activities (n = 24, one-way ANOVA), p_MsrB1 < 0.005 and p_MsrA > 0.1.

FIG. 5.

Regulation of MsrB1 and MsrA expression and MsrB and MsrA activities in HET outbred mice. (A) Female mice were subjected to six indicated diets (four animals per diet). Their liver samples were analyzed by Western blotting, as indicated. The proteins probed are indicated on the left and protein loading is shown by Amido Black staining on bottom image. Analysis of MsrB (B) and MsrA (C) activities (n = 24, one-way ANOVA), p_MsrB1 < 0.005 and p_MsrA > 0.1.

FIG. 6.

Comparison of MsrB and MsrA activities in different mouse strains. 10-month-old Balb/c (10m), 30 month-old Balb/c (30m), calorie restriction (CR) group, TGFα transgenic, TGFα/i6A^- double transgenic, and HET outbred mice were maintained on a 0.4 ppm Se diet, and MsrB (A) and MsrA (B) activities determined as shown.