Ablation of Selenbp1 Alters Lipid Metabolism via the Pparα Pathway in Mouse Kidney

Abstract

Selenium-binding protein 1 (Selenbp1) is a 2,3,7,8-tetrechlorodibenzo-p-dioxin inducible protein whose function is yet to be comprehensively elucidated. As the highly homologous isoform, Selenbp2, is expressed at low levels in the kidney, it is worthwhile comparing wild-type C57BL mice and Selenbp1-deficient mice under dioxin-free conditions. Accordingly, we conducted a mouse metabolomics analysis under non-dioxin-treated conditions. DNA microarray analysis was performed based on observed changes in lipid metabolism-related factors. The results showed fluctuations in the expression of numerous genes. Real-time RT-PCR confirmed the decreased expression levels of the cytochrome P450 4a (Cyp4a) subfamily, known to be involved in fatty acid ω- and ω-1 hydroxylation. Furthermore, peroxisome proliferator-activated receptor-α (Pparα) and retinoid-X-receptor-α (Rxrα), which form a heterodimer with Pparα to promote gene expression, were simultaneously reduced. This indicated that reduced Cyp4a expression was mediated via decreased Pparα and Rxrα. In line with this finding, increased levels of leukotrienes and prostaglandins were detected. Conversely, decreased hydrogen peroxide levels and reduced superoxide dismutase (SOD) activity supported the suppression of the renal expression of Sod1 and Sod2 in Selenbp1-deficient mice. Therefore, we infer that ablation of Selenbp1 elicits oxidative stress caused by increased levels of superoxide anions, which alters lipid metabolism via the Pparα pathway.

Keywords: Ppar; kidney; lipid metabolism; mouse; oxidative stress; peroxisome proliferator-activated receptor-alpha; selenium binding protein 1.

Figure 1

Effect of fasting on the mRNA expression of Selenbp1 and Selenbp2 (A) and protein expression levels of Selenbp (B,C) in the kidney of wild-type and Selenbp1-KO mice. A. The kidneys of 8-week-old male mice were removed. The relative mRNA level was normalized to that of β-actin. Values represent the mean ± standard error of the mean (SEM). Non-fasting (n = 6); fasting (n = 6). Significant differences from the non-fasting group: ** p < 0.01; *** p < 0.001. B. The kidneys of 8-week-old male wild-type and Selenbp1-KO mice were removed. Individual kidney S9-fractions (20 μg protein) prepared from wild-type and Selenbp1-KO mice were subjected to SDS-PAGE (10% separation gel). Selenbp was detected using a mouse anti-human Selenbp1 antibody. β-actin was detected using a mouse anti-β-actin antibody. Lanes 1–2, WT non-fasting; 3–4, WT fasting; 5–6, Selenbp1-KO non-fasting; 7–8, Selenbp1-KO fasting. Representative data are presented. Two of the five randomly selected samples were analyzed. C. Values represent the mean ± SEM. Non-fasting (n = 5); fasting (n = 5). N.D., not detectable; WT, wild-type; KO, knockout; Selenbp1/2, selenium-binding protein 1/2.

Figure 2

Changes in the renal metabolomic profile of Selenbp1-KO mice compared with the control group using UPLC-TOF/MS. Principal component analysis (PCA) of the effect of Selenbp1 deletion on the kidney metabolome: data from positive (A) and negative (B) ion mode analysis. Selenbp1 deletion affects the profile of the kidney metabolome. Each dot represents a different animal (n = 11 mice per group). Wild-type and Selenbp1-KO mice are shown in black and red, respectively. S-plot based on the PCA regarding the effect of Selenbp1 deletion on the kidney metabolome: data from positive (C) and negative (D) ion mode analysis. Fragment ions in UPLC-TOF-MS analysis are altered by Selenbp1 deletion in the kidneys of male mice (8 weeks old). Each dot represents a single ion with a specific mass (m/z). The criteria for selecting ions significantly altered by Selenbp1 deletion were set either at more than +0.8 or less than −0.8 of the correlation coefficient (red dots). Conditions were under 20 h of fasting. Selenbp1, selenium-binding protein 1; UPLC-TOF/MS, ultra-performance liquid chromatography-time-of-flight mass spectrometry; KO, knockout.

Figure 3

Heatmap of renal mRNAs significantly altered in male adult mice (8 weeks old) by Selenbp1 deletion. Genes whose expression was significantly increased or decreased by Selenbp1 deletion are shown in red and green, respectively. For the magnitude of the alteration, see the color gradation shown in the figure. Each lane is the mRNA prepared from male mice at 8 weeks. Eighteen genes, indicated by arrows and numbers, are shown in Table 2. Conditions under 20 h of fasting. Selenbp1, Selenium-binding protein 1.

Figure 4

Effect of Selenbp1 ablation on the renal expression of lipid metabolism-related enzymes. The kidneys were collected from 8-week-old male mice fasted for 20 h. The relative mRNA levels of the indicated enzymes were analyzed by real-time RT-PCR and normalized to β-actin mRNA. Each bar represents the mean ± standard error of the mean (SEM) of six mice. β-Actin was used as an internal control. * p < 0.05, ** p < 0.01. Selenbp1, selenium-binding protein 1; RT-PCR, reverse transcription-polymerase chain reaction.

Figure 5

Effects of Selenbp1 on the renal redox reaction. (A). Effect of Selenbp1 ablation on the renal expression of oxidative stress-related enzymes. (B). SOD activity and hydrogen peroxide concentrations in mice kidneys. SOD activity was determined by a WST1 assay. Hydrogen peroxide concentration was determined by a quantitative peroxide assay. The kidneys were isolated from 8-week-old male mice who were fasted for 20 h. Each bar represents the mean ± standard error of the mean (SEM) of five mice. * p < 0.05; *** p < 0.001. (C). TBARS used as the index of renal lipid peroxidation in Selenbp1-deficient mice. Each bar represents the mean ± SEM of 10 mice. Selenbp1, selenium binding protein 1; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substance.

Figure 6

Serum and kidney levels of selenium (µg/g) in wild-type and Selenbp1-KO mice by Agilent Technologies ICP-MS 7700× detection. Each bar represents the mean ± standard error of the mean (SEM) of three mice performed in triplicate: (A) kidney; (B) serum. Details are described in the Materials and Methods section. Selenbp1, selenium binding protein 1; ICP-MS, inductively coupled plasma mass spectrometry.