Loss of housekeeping selenoprotein expression in mouse liver modulates lipoprotein metabolism

Abstract

Selenium is incorporated into proteins as selenocysteine (Sec), which is dependent on its specific tRNA, designated tRNA([Ser]Sec). Targeted removal of the tRNA([Ser]Sec) gene (Trsp) in mouse hepatocytes previously demonstrated the importance of selenoproteins in liver function. Herein, analysis of plasma proteins in this Trsp knockout mouse revealed increases in apolipoprotein E (ApoE) that was accompanied by elevated plasma cholesterol levels. The expression of genes involved in cholesterol biosynthesis, metabolism and transport were also altered in knockout mice. Additionally, in two transgenic Trsp mutant mouse lines (wherein only housekeeping selenoprotein synthesis was restored), the expression of ApoE, as well as genes involved in cholesterol biosynthesis, metabolism and transport were similar to those observed in wild type mice. These data correlate with reports that selenium deficiency results in increased levels of ApoE, indicating for the first time that housekeeping selenoproteins have a role in regulating lipoprotein biosynthesis and metabolism.

Fig. 1

Analysis of plasma proteins in wild type and ΔTrsp mice. (A) Plasma proteins from wild type and ΔTrsp mice were electrophoresed and stained with Coomassie Blue. The elevated protein (~35 kDa) in ΔTrsp mice is indicated by an arrow. (B) Mouse ApoE sequence. Peptides detected by MS/MS analysis are underlined and shown in bold.

Fig. 2

Analysis of ApoE and Apoe mRNA in wild type and ΔTrsp mice. (A) Coomassie Blue staining of plasma proteins from male and female wild type and ΔTrsp mice (upper panel) and immunodetection of ApoE using polyclonal anti-ApoE antibodies in plasma from the same mice (lower panel). (B) The relative Apoe mRNA levels in liver and kidney samples of wild type and ΔTrsp mice, determined by real-time PCR. The level of Apoe mRNA in each sample was normalized to that of 18S rRNA and the normalized value for Apoe mRNA in ΔTrsp mice was then plotted relative to that of wild type mice along with the error bars. The results are representations of 4 independent experiments, each carried out in triplicate (*, p < 0.0005 versus wild type mice).

Fig. 3

Cholesterol levels and analysis of genes involved in cholesterol metabolism in wild type and ΔTrsp mice. (A) Examination of plasma cholesterol levels in wild type and ΔTrsp male and female mice. The values represent means ± SE, n = 5 mice/sex · genotype⁻¹ (p < 0.01 for both genders). (B) Microarray analysis, using total RNA from liver of wild type and ΔTrsp mice showing genes with altered expression involved in cholesterol biosynthesis, metabolism or transport. Results from four independent hybridizations are shown in the column designated ΔFOLD wherein an arrow pointing up indicates a relative increase, and an arrow pointing down, a relative decrease in mRNA expression; and the standard error is shown in the column designated SE. (C) Real-time PCR analysis of mRNA levels of some of the genes tabulated in Fig. 3B. The results represent 3–4 independent experiments, each carried out in triplicate and shown along with the error bars (*, p < 0.005; **, p < 0.1)

Fig. 4

ApoE and cholesterol levels in A34 and G37 mice. ApoE was assessed by western blotting and cholesterol levels were measured in plasma lipids of wild type, ΔTrsp, A34 and G37 mice. (A) Coomassie Blue staining of plasma proteins from wild type, ΔTrsp, A34 and G37 male mice (upper panel) and western blotting carried out using polyclonal anti-ApoE antibodies (lower panel). (B) Plasma cholesterol levels. Values represent means ± SE, n = 5 mice/genotype for wild type and ΔTrsp mice and n = 4 mice/genotype for A34 and G37 mice (*, p < 0.01).